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The clinical handling of chronic virus infections remains a challenge. Here we describe recent progress in the understanding of virus - host interaction dynamics. Based on the systems biology concept of multi-stability and the prediction of multiplicative cooperativity between virus-specific cytotoxic T cells and neutralising antibodies, we argue for the requirements to engage multiple immune system components for functional cure strategies. Our arguments are derived from LCMV model system studies and are translated to HIV-1 infection. The authors are supported by the Russian Science Foundation (RSF grant no. 18-11-00171), the Russian Foundation of Basic Research (RFBR grant no. 20-01-00352), “la Caixa” Foundation under the project code HR17-00199, the Spanish Ministry of Science and Innovation grant no. PID2019-106323RB-I00 AEI//10.13039/501100011033, and “Unidad de Excelencia María de Maeztu”, funded by the MCIN and the AEI (DOI: 10.13039/501100011033) Ref: CEX2018-000792-M.

Individuals infected with the human immunodeficiency virus type 1 (HIV-1) belong to the group of people most vulnerable to SARS-CoV-2 infections and the associated disease COVID-19. Here we describe SARS-CoV-2-specific antibody and cellular immune responses in a small cohort of immunological non-responder HIV-1 patients (HIV-INRs) after receiving the COVID-19 mRNA-based BioNTech/Pfizer vaccine. Compared to the control group of vaccinated healthy individuals that all developed a virus-specific immune response, 5 of 10 vaccinated HIV-1 patients showed insufficient immune responses. The lack of response was not directly correlated with patients CD4 cell counts. Three of the five non-responders that agreed to receive a booster vaccination subsequently generated a virus-specific response. Thus, even HIV-INRs can be efficiently vaccinated against COVID-19 but may require a follow-up by virus-specific immune monitoring to guarantee clinical vaccine benefits. The authors are supported by Spanish Melanoma Group Grant (GEM) (III Beca GEM para Grupos Emergentes), the Spanish Ministry of Science and Innovation grant no. PID2019-106323RB-I00 AEI//10.13039/501100011033, the “Unidad de Excelencia María de Maeztu”, funded by the MCIN and the AEI (DOI: 10.13039/501100011033); Ref: CEX2018-000792-M and by a FIS Project “PI19/00019” funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union.

Cancer patients (CPs) have been identified as particularly vulnerable to SARS-CoV-2 infection, and therefore are a priority group for receiving COVID-19 vaccination. From the patients with advanced solid tumors, about 20% respond very efficiently to immunotherapy with anti-PD1/PD-L1 antibodies and achieve long lasting cancer responses. It is unclear whether an efficient cancer-specific immune response may also correlate with an efficient response upon COVID-19 vaccination. Here, we explored the antiviral immune response to the mRNA-based COVID-19 vaccine BNT162b2 in a group of 11 long-lasting cancer immunotherapy responders. We analysed the development of SARS-CoV-2-specific IgG serum antibodies, virus neutralizing capacities and T cell responses. Control groups included patients treated with adjuvant cancer immunotherapy (IMT, cohort B), CPs not treated with immunotherapy (no-IMT, cohort C) and healthy controls (cohort A). The median ELISA IgG titers significantly increased after the prime-boost COVID vaccine regimen in all cohorts (Cohort A: pre-vaccine = 900 (100-2700), 3 weeks (w) post-boost = 24300 (2700-72900); Cohort B: pre-vaccine = 300 (100-2700), 3 w post-boost = 8100 (300-72900); Cohort C: pre-vaccine = 500 (100-2700), 3 w post-boost = 24300 (300-72900)). However, at the 3 w post-prime time-point, only the healthy control group showed a statistically significant increase in antibody levels (Cohort A = 8100 (900-8100); Cohort B = 900 (300-8100); Cohort C = 900 (300-8100)) (P < 0.05). Strikingly, while all healthy controls generated high-level antibody responses after the complete prime-boost regimen (Cohort A = 15/15 (100%), not all CPs behaved alike [Cohort B= 12/14 (84'6%); Cohort C= 5/6 (83%)]. Their responses, including those of the long-lasting immunotherapy responders, were more variable (Cohort A: 3 w post-boost (median nAb titers = 95.32 (84.09-96.93), median Spike-specific IFN-γ response = 64 (24-150); Cohort B: 3 w post-boost (median nAb titers = 85.62 (8.22-97.19), median Spike-specific IFN-γ response (28 (1-372); Cohort C: 3 w post-boost (median nAb titers = 95.87 (11.8-97.3), median Spike-specific IFN-γ response = 67 (20-84)). Two long-lasting cancer responders did not respond properly to the prime-boost vaccination and did not generate S-specific IgGs, neutralizing antibodies or virus-specific T cells, although their cancer immune control persisted for years. Thus, although mRNA-based vaccines can induce both antibody and T cell responses in CPs, the immune response to COVID vaccination is independent of the capacity to develop an efficient anti-cancer immune response to anti PD-1/PD-L1 antibodies. The authors are supported by Spanish Melanoma Group Grant (III Beca GEM para Grupos Emergentes), the Spanish Ministry of Science and Innovation grant no. PID2019-106323RB-I00 AEI//10.13039/501100011033, the “Unidad de Excelencia María de Maeztu”, funded by the MCIN and the AEI (DOI: 10.13039/501100011033); Ref: CEX2018-000792-M.

A growing number of studies indicate that mRNAs and long ncRNAs can affect protein populations by assembling dynamic ribonucleoprotein (RNP) granules. These phase-separated molecular 'sponges', stabilized by quinary (transient and weak) interactions, control proteins involved in numerous biological functions. Retroviruses such as HIV-1 form by self-assembly when their genomic RNA (gRNA) traps Gag and GagPol polyprotein precursors. Infectivity requires extracellular budding of the particle followed by maturation, an ordered processing of ∼2400 Gag and ∼120 GagPol by the viral protease (PR). This leads to a condensed gRNA-NCp7 nucleocapsid and a CAp24-self-assembled capsid surrounding the RNP. The choreography by which all of these components dynamically interact during virus maturation is one of the missing milestones to fully depict the HIV life cycle. Here, we describe how HIV-1 has evolved a dynamic RNP granule with successive weak-strong-moderate quinary NC-gRNA networks during the sequential processing of the GagNC domain. We also reveal two palindromic RNA-binding triads on NC, KxxFxxQ and QxxFxxK, that provide quinary NC-gRNA interactions. Consequently, the nucleocapsid complex appears properly aggregated for capsid reassembly and reverse transcription, mandatory processes for viral infectivity. We show that PR is sequestered within this RNP and drives its maturation/condensation within minutes, this process being most effective at the end of budding. We anticipate such findings will stimulate further investigations of quinary interactions and emergent mechanisms in crowded environments throughout the wide and growing array of RNP granules. This work was supported in part by the European Project THINPAD “Targeting the HIV-1 Nucleocapsid Protein to fight Antiretroviral Drug Resistance” (FP7-Grant Agreement 601969), by Foundation Clinic, by ANRS, by SIDACTION, and with federal funds from the NCI/NIH, under Contract No. HHSN261200800001E with Leidos Biomedical Research, Inc. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government (R.J.G.). S.L. acknowledges funding by the Marie-Curie IEF fellowship (FP7-Grant Agreement 237738) and is grateful to Maria Solà (IBMB-CSIC). S.K.S. and A.M. acknowledge support from amfAR Mathilde Krim Fellowship in Basic Biomedical Research number 108680 and the Spanish Ministry of Economy and Competitiveness and FEDER (Grant no. SAF2013-46077-R). S.K.S. also gratefully acknowledges support from the Volkswagen Foundation “Experiment! Funding Initiative” grant number 93874 and from the Klaus Tschira Stiftung.

SARS-CoV-2 infection represents a global threat to human health. Various approaches were employed to reveal the pathogenetic mechanisms of COVID-19. Mathematical and computational modelling is a powerful tool to describe and analyze the infection dynamics in relation to a plethora of processes contributing to the observed disease phenotypes. In our study here, we formulate and calibrate a deterministic model of the SARS-CoV-2 life cycle. It provides a kinetic description of the major replication stages of SARS-CoV-2. Sensitivity analysis of the net viral progeny with respect to model parameters enables the identification of the life cycle stages that have the strongest impact on viral replication. These three most influential parameters are (i) degradation rate of positive sense vRNAs in cytoplasm (negative effect), (ii) threshold number of non-structural proteins enhancing vRNA transcription (negative effect), and (iii) translation rate of non-structural proteins (positive effect). The results of our analysis could be used for guiding the search for antiviral drug targets to combat SARS-CoV-2 infection. The reported study was funded by RFBR according to the research project numbers 20-04-60157 and 20-01-00352. AM is also supported by the Spanish Ministry of Science and Innovation grant no. PID2019-106323RB-I00(AEI/MINEICO/FEDER, UE), and “Unidad de Excelencia María de Maeztu”, funded by the AEI (CEX2018-000792-M). G.B. and D.G. were partly supported by Moscow Center for Fundamental and Applied Mathematics (agreement with the Ministry of Education and Science of the Russian Federation No. 075-15-2019-1624).

info:eu-repo/semantics/publishedVersion

Mathematical modelling of infection processes in cells is of fundamental interest. It helps to understand the SARS-CoV-2 dynamics in detail and can be useful to define the vulnerability steps targeted by antiviral treatments. We previously developed a deterministic mathematical model of the SARS-CoV-2 life cycle in a single cell. Despite answering many questions, it certainly cannot accurately account for the stochastic nature of an infection process caused by natural fluctuation in reaction kinetics and the small abundance of participating components in a single cell. In the present work, this deterministic model is transformed into a stochastic one based on a Markov Chain Monte Carlo (MCMC) method. This model is employed to compute statistical characteristics of the SARS-CoV-2 life cycle including the probability for a non-degenerate infection process. Varying parameters of the model enables us to unveil the inhibitory effects of IFN and the effects of the ACE2 binding affinity. The simulation results show that the type I IFN response has a very strong effect on inhibition of the total viral progeny whereas the effect of a 10-fold variation of the binding rate to ACE2 turns out to be negligible for the probability of infection and viral production. This research was funded by the Russian Science Foundation (grant number 18-11-00171) and partly by the Russian Foundation for Basic Research according to the research project numbers 20-04-60157 and 20-01-00352. A.M. is also supported by the Spanish Ministry of Science and Innovation grant no. PID2019-106323RB-I00(AEI/MINEICO/FEDER, UE) and “Unidad de Excelencia María de Maeztu” funded by the AEI (CEX2018-000792-M). D.G. was partly supported by the Moscow Center for Fundamental and Applied Mathematics (agreement with the Ministry of Education and Science of the Russian Federation No. 075-15-2019-1624).

Background Moving from the molecular and cellular level to a multi-scale systems understanding of immune responses requires the development of novel approaches to integrate knowledge and data from different biological levels into mechanism-based integrative mathematical models. The aim of our study is to present a methodology for a hybrid modelling of immunological processes in their spatial context. Methods A two-level hybrid mathematical model of immune cell migration and interaction integrating cellular and organ levels of regulation for a 2D spatial consideration of idealized secondary lymphoid organs is developed. It considers the population dynamics of antigen-presenting cells, CD4 + and CD8 + T lymphocytes in naive-, proliferation- and differentiated states. Cell division is assumed to be asymmetric and regulated by the extracellular concentration of interleukin-2 (IL-2) and type I interferon (IFN), together controlling the balance between proliferation and differentiation. The cytokine dynamics is described by reaction-diffusion PDEs whereas the intracellular regulation is modelled with a system of ODEs. Results The mathematical model has been developed, calibrated and numerically implemented to study various scenarios in the regulation of T cell immune responses to infection, in particular the change in the diffusion coefficient of type I IFN as compared to IL-2. We have shown that a hybrid modelling approach provides an efficient tool to describe and analyze the interplay between spatio-temporal processes in the emergence of abnormal immune response dynamics. Discussion Virus persistence in humans is often associated with an exhaustion of T lymphocytes. Many factors can contribute to the development of exhaustion. One of them is associated with a shift from a normal clonal expansion pathway to an altered one characterized by an early terminal differentiation of T cells. We propose that an altered T cell differentiation and proliferation sequence can naturally result from a spatial separation of the signaling events delivered via TCR, IL-2 and type I IFN receptors. Indeed, the spatial overlap of the concentration fields of extracellular IL-2 and IFN in lymph nodes changes dynamically due to different migration patterns of APCs and CD4 + T cells secreting them. Conclusions The proposed hybrid mathematical model of the immune response represents a novel analytical tool to examine challenging issues in the spatio-temporal regulation of cell growth and differentiation, in particular the effect of timing and location of activation signals. Electronic supplementary material The online version of this article (doi:10.1186/s12865-017-0205-0) contains supplementary material, which is available to authorized users.

Based on the structure of an HIV-1 entry inhibitor peptide two stapled- and a retro-enantio peptides have been designed to provide novel prevention interventions against HIV transmission. The three peptides show greater inhibitory potencies in cellular and mucosal tissue pre-clinical models than the parent sequence and the retro-enantio shows a strengthened proteolytic stability. Since HIV-1 fusion inhibitor peptides need to be embedded in the membrane to properly interact with their viral target, the structural features were determined by NMR spectroscopy in micelles and solved by using restrained molecular dynamics calculations. Both parent and retro-enantio peptides demonstrate a topology compatible with a shared helix–turn–helix conformation and assemble similarly in the membrane maintaining the active conformation needed for its interaction with the viral target site. This study represents a straightforward approach to design new targeted peptides as HIV-1 fusion inhibitors and lead us to define a retro-enantio peptide as a good candidate for pre-exposure prophylaxis against HIV-1., Financial support from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and the European Regional Development Fund (Grant RTI2018-094120-B-I00 to I.H.) is gratefully acknowledged. A.M. is supported by a grant SAF2016-75505-R (AEI/MINECO/FEDER, UE) and through the “María de Maeztu” Programme for Units of Excellence in R&D (MDM-2014-0370). The 500-MHz spectrometer was purchased in part through a Research Infraestructure MINECO-FEDER fund (Grant CSIC13-4E-2076). Partial financial support from the Spanish National Research Council (CSIC) is also gratefully acknowledged.

IMPORTANCE: Therapies targeting the programmed cell death 1 (PD-1) receptor or its ligand (PD-L1), such as the humanized monoclonal antibody durvalumab, have shown durable clinical responses in several tumor types. However, concerns about the safety and feasibility of PD-1/PD-L1 blockade in HIV-1-infected individuals have led to the exclusion of these patients from clinical trials on cancer immunotherapies. OBJECTIVE: To evaluate the feasibility and safety of durvalumab treatment in patients with advanced cancer and virologically controlled HIV-1 infection. DESIGN, SETTING, AND PARTICIPANTS: The DURVAST study was a nonrandomized, open-label, phase 2 clinical trial in patients with any solid tumor type in which anti-PD-1 or anti-PD-L1 antibodies have approved indications or for which there are data of antitumoral activity with no other available curative therapy. All patients had basal undetectable plasma viremia while undergoing combination antiretroviral therapy. INTERVENTIONS: Treatment consisted of intravenous infusion of durvalumab (1500 mg every 4 weeks) until disease progression or unacceptable toxic effects. MAIN OUTCOMES AND MEASURES: Adverse events were graded with the use of the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.03. Tumor response was evaluated using the Response Evaluation Criteria in Solid Tumors version 1.1. RESULTS: A total of 20 HIV-1-infected patients with advanced cancer were enrolled; 16 (80%) were male, the median (range) age was 54 (30-73) years, and 12 (60%) had progressed with previous cancer treatment lines. A median (range) of 4 (1-16) cycles of durvalumab were administered. Drug-related adverse events were observed in 50% of patients, and all were grade 1 and 2 (mainly diarrhea, asthenia, and arthromyalgia). Four of 16 response-evaluable patients (25%) had a partial response. Five patients (31%) had stable disease, including 4 with durable stable disease (disease control rate of 50%). CD4+ and CD8+ T-cell counts and plasma HIV-1 viremia remained stable throughout the study. CONCLUSIONS AND RELEVANCE: Durvalumab treatment was feasible and safe in HIV-1-infected patients with cancer receiving combination antiretroviral therapy. HIV-1-infected patients on suppressive antiretroviral therapy with advanced cancer should have access to cancer immunotherapy treatments. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03094286.

We advocate the widespread use of UV-C light as a short-term, easily deployable, and affordable way to limit virus spread in the current SARS-CoV-2 pandemic. Radical social distancing with the associated shutdown of schools, restaurants, sport clubs, workplaces, and traveling has been shown to be effective in reducing virus spread, but its economic and social costs are unsustainable in the medium term. Simple measures like frequent handwashing, facial masks, and other physical barriers are being commonly adopted to prevent virus transmission. However, their efficacy may be limited, particularly in shared indoor spaces, where, in addition to airborne transmission, elements with small surface areas such as elevator buttons, door handles, and handrails are frequently used and can also mediate transmission. We argue that additional measures are necessary to reduce virus transmission when people resume attending schools and jobs that require proximity or some degree of physical contact. Among the available alternatives, UV-C light satisfies the requirements of rapid, widespread, and economically viable deployment. Its implementation is only limited by current production capacities, an increase of which requires swift intervention by industry and authorities.

The surveillance of host body tissues by immune cells is central for mediating their defense function. In vivo imaging technologies have been used to quantitatively characterize target cell scanning and migration of lymphocytes within lymph nodes (LNs). The translation of these quantitative insights into a predictive understanding of immune system functioning in response to various perturbations critically depends on computational tools linking the individual immune cell properties with the emergent behavior of the immune system. By choosing the Newtonian second law for the governing equations, we developed a broadly applicable mathematical model linking individual and coordinated T-cell behaviors. The spatial cell dynamics is described by a superposition of autonomous locomotion, intercellular interaction, and viscous damping processes. The model is calibrated using in vivo data on T-cell motility metrics in LNs such as the translational speeds, turning angle speeds, and meandering indices. The model is applied to predict the impact of T-cell motility on protection against HIV infection, i.e., to estimate the threshold frequency of HIV-specific cytotoxic T cells (CTLs) that is required to detect productively infected cells before the release of viral particles starts. With this, it provides guidance for HIV vaccine studies allowing for the migration of cells in fibrotic LNs. This work is supported by the Russian Science Foundation (grant 18-11-00171). VV was supported by a grant from the RUDN University Program 5-100. AM is also supported by a grant from the Spanish Ministry of Economy, Industry and Competitiveness and FEDER grant no. SAF2016-75505-R (AEI/MINEICO/FEDER, UE) and the María de Maeztu Programme for Units of Excellence in R&D (MDM-2014-0370).

The modern era of research in immunology is characterized by an unprecedented level of detail about structural characteristics of the immune system and the regulation of activities of its numerous components, which function together as a whole distributed-parameter system. Mathematical modeling provides an analytical tool to describe, analyze, and predict the dynamics of immune responses by applying a reductionist approach. In modern systems immunology and mathematical immunology as a new interdisciplinary field, a great challenge is to formulate the mathematical models of the human immune system that reflect the level achieved in understanding its structure and describe the processes that sustain its function. To this end, a systematic development of multiscale mathematical models has to be advanced. An appropriate methodology should consider (1) the intracellular processes of immune cell fate regulation, (2) the population dynamics of immune cells in various organs, and (3) systemic immunophysiological processes in the whole host organism. Main studies aimed at modeling the intracellular regulatory networks are reviewed in the context of multiscale mathematical modelling. The processes considered determine the regulation of the immune cell fate, including activation, division, differentiation, apoptosis, and migration. Because of the complexity and high dimensionality of the regulatory networks, identifying the parsimonious descriptions of signaling pathways and regulatory loops is a pressing problem of modern mathematical immunology.

International audience; Spreading of viral infection in the tissues such as lymph nodes or spleen depends on virus multiplication in the host cells, their transport and on the immune response. Reaction-diffusion systems of equations with delays in cell proliferation and death by apoptosis represent an appropriate model to study this process. The properties of the cells of the immune system and the initial viral load determine the spatiotemporal regimes of infection spreading. Infection can be completely eliminated or it can persist at some level together with a certain chronic immune response in a spatially uniform or oscillatory mode. Finally, the immune cells can be completely exhausted leading to a high viral load persistence in the tissue. It has been found experimentally, that virus proteins can affect the immune cell migration. Our study shows that both the motility of immune cells and the virus infection spreading represented by the diffusion rate coefficients are relevant control parameters determining the fate of virus-host interaction.

Cancer immunotherapy with antibodies against immune checkpoints has made impressive advances in the last several years. The most relevant drugs target programmed cell death 1 (PD-1) expressed on T cells or its ligand, the programmed cell death ligand 1 (PD-L1), expressed on cancer cells, and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). Unfortunately, cancer patients with HIV infection are usually excluded from cancer clinical trials, because there are concerns about the safety and the anti-tumoral activity of these novel therapies in patients with HIV infection. Several retrospective studies and some case reports now support the notion that antibodies against immune checkpoints are safe and active in cancer patients with HIV infection, but prospective data in these patients are lacking. In addition, signs of antiviral activity with increase in CD4 T cell counts, plasma viremia reduction or decrease in the viral reservoir have been reported in some of the patients treated, although no patient achieved a complete clearance of the viral reservoir. Here we briefly summarize all clinical cases reported in the literature, as well as ongoing clinical trials testing novel immunotherapy drugs in cancer patients with HIV infection.

Lack of effective strategies for killing cells latently infected with HIV-1 limits the eradication of AIDS. Unfortunately, current antiretroviral inhibitors are designed to target virus production but not latent infection. Interestingly, some non-nucleoside reverse transcriptase inhibitors (NNRTIs) have shown off-design effects, specifically, premature activation of HIV-1 protease (PR) within virus-infected cells that induces apoptosis. Here, we analyze an equilibrium model of HIV-1 reverse transcriptase (RT) binding to NNRTIs to understand the optimal binding characteristics that enhance RT dimerization within embedded GagPol dimers. This would allow NNRTIs to act as PR autoactivation enhancers (PAEs). We compute that ∼700-fold enhancement is theoretically possible by PAEs. Both a strong drug-dimer binding affinity (KD12 10) are required for significant enhancement (∼50-fold or more) relative to the drug-free dimer concentration within a drug concentration limit of 10 μM. Our approach rationalizes the observed effects of efavirenz on premature activation of PR and may be useful to guide the design of suitable drug candidates and their optimal dosage regimens for this therapy class.

BACKGROUND & AIMS: Soraphen A (SorA) is a myxobacterial metabolite that inhibits the acetyl-CoA carboxylase, a key enzyme in lipid biosynthesis. We have previously identified SorA to efficiently inhibit the human immunodeficiency virus (HIV). The aim of the present study was to evaluate the capacity of SorA and analogues to inhibit hepatitis C virus (HCV) infection. METHODS: SorA inhibition capacity was evaluated in vitro using cell culture derived HCV, HCV pseudoparticles and subgenomic replicons. Infection studies were performed in the hepatoma cell line HuH7/Scr and in primary human hepatocytes. The effects of SorA on membranous web formation were analysed by electron microscopy./nRESULTS:/nSorA potently inhibits HCV infection at nanomolar concentrations. Obtained EC50 values were 0.70 nM with a HCV reporter genome, 2.30 nM with wild-type HCV and 2.52 nM with subgenomic HCV replicons. SorA neither inhibited HCV RNA translation nor HCV entry, as demonstrated with subgenomic HCV replicons and HCV pseudoparticles, suggesting an effect on HCV replication. Consistent with this, evidence was obtained that SorA interferes with formation of the membranous web, the site of HCV replication. Finally, a series of natural and synthetic SorA analogues helped to establish a first structure-activity relationship./nCONCLUSIONS:/nSorA has a very potent anti-HCV activity. Since it also interferes with the membranous web formation, SorA is an excellent tool to unravel the mechanism of HCV replication. The authors are supported by grants BFU2013-44629-R (to JD, GK and GPV), SAF2013-46077-R (to/nAM and JM), both from the Spanish Ministry of Economy and Competitiveness and FEDER, and from the German Centre for Infection Research (DZIF, to MB and RM)

Soraphen A is a myxobacterial metabolite that blocks the acetyl-coenzyme A carboxylase of the host and was previously identified as a novel HIV inhibitor. Here, we report that soraphen A acts by reducing virus production and altering the gp120 virion content, impacting entry capacity and infectivity. These effects are partially reversed by addition of palmitic acid, suggesting that inhibition of HIV envelope palmitoylation is one of the mechanisms of antiviral action. The research was funded by the German Centre for Infection Research (DZIF), Spanish Ministry of Economy, Industry and Competitiveness and FEDER BES-2011-048569, Spanish Ministry of Economy, Industry and Competitiveness and FEDER SAF2013-46077-R SAF2016-75505-R, the Spanish Ministry of Economy, Industry and Competitiveness and FEDER BFU2016-80039-R and the Maria de Maetzu Programme for Units of Excellence in R&DMDM-2014-0370

A hepatitis C virus (HCV) epidemic affecting HIV-infected men who have sex with men (MSM) is expanding worldwide. In spite of the improved cure rates obtained with the new direct-acting antiviral drug (DAA) combinations, the high rate of reinfection within this population calls urgently for novel preventive interventions. In this study, we determined in cell culture and ex vivo experiments with human colorectal tissue that lipoquads, G-quadruplex DNA structures fused to cholesterol, are efficient HCV pangenotypic entry and cell-to-cell transmission inhibitors. Thus, lipoquads may be promising candidates for the development of rectally applied gels to prevent HCV transmission., We thank Paul Ziprin and the Department of Surgery and Cancer at St. Mary’s Hospital, Imperial College London, for their assistance in obtaining human tissue. We do not have anything to disclose regarding funding or conflicts of interest with respect to the manuscript. We are supported by grants BFU2013-44629-R (to J.D., G.K., G.P.- V., and A.P.D.L.) and BFU2016-80039-R (to J.D. and G.P.-V.) and also through the María de Maeztu Programme for Units of Excellence in R&D (MDM-2014-0370), all from the Spanish Ministry of Economy, Industry and Competitiveness and FEDER. Work at Imperial College London (C.H.) was funded by EC FP7 grant 305578 (PathCO).

Processing bodies (P-bodies) are cytoplasmatic mRNP granules containing non-translating mRNAs and proteins from the mRNA decay and silencing machineries. The mechanism of P-body assembly has been typically addressed by depleting P-body components. Here we apply a complementary approach and establish an automated cell-based assay platform to screen for molecules affecting P-body assembly. From a unique library of compounds derived from myxobacteria, 30 specifically inhibited P-body assembly. Gephyronic acid A (GA), a eukaryotic protein synthesis inhibitor, showed the strongest effect. GA also inhibited, under stress conditions, phosphorylation of eIF2α and stress granule formation. Other hits uncovered interesting novel links between P-body assembly, lipid metabolism, and internal organelle physiology. The obtained results provide a chemical toolbox to manipulate P-body assembly and function.

Single nucleotide polymorphisms (SNPs) located in the chromosome region 17q12-q21 are risk factors for asthma. Particularly, there are cis-regulatory haplotypes within this region that regulate differentially the expression levels of ORMDL3, GSDMB and ZPBP2 genes. Remarkably, ORMDL3 has been shown to modulate lymphocyte activation parameters in a heterologous expression system. In this context, it has been shown that Th2 and Th17 cytokine production is affected by SNPs in this region. Therefore, we aim to assess the impact of hereditary components within region 17q12-q21 on the activation profile of human T lymphocytes, focusing on the haplotype formed by allelic variants of SNPs rs7216389 and rs12936231. We measured calcium influx and activation markers, as well as the proliferation rate upon T cell activation. Haplotype-dependent differences in mRNA expression levels of IL-2 and INF-γ were observed at early times after activation. In addition, the allelic variants of these SNPs impacted on the extent of calcium influx in resting lymphocytes and altered proliferation rates in a dose dependent manner. As a result, the asthma risk haplotype carriers showed a lower threshold of saturation during activation. Finally, we confirmed differences in activation marker expression by flow cytometry using phytohemagglutinin, a strong polyclonal stimulus. Altogether, our data suggest that the genetic component of pro-inflammatory pathologies present in this chromosome region could be explained by different T lymphocyte activation dynamics depending on individual allelic heredity. This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (SAF2010-16725, SAF2013-46077-R, SAF2014-52228-R, BES-2011-043839), Fondo de Investigación Sanitaria (Red HERACLES RD12/0042/0014), Fundació la Marató de TV3 (20134030) and Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT 3150113). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

OBJECTIVE To assess the effect of antigen-exposure on the T-cell repertoire in the chronic phase of HIV-infection. DESIGN This is a prospective cross-sectional study. METHODS HIV-seropositive patients and immunocompetent controls from tuberculosis low and high-endemic countries were recruited. Mycobacterium tuberculosis (purified protein derivative; PPD)-specific CD4 T-cell responses were quantified directly from whole blood using flow-cytometric analysis of intracellular cytokines after specific stimulation. T-cell reactivity toward cytomegalovirus (CMV) or Staphylococcus aureus Enterotoxin B (SEB) served as control. RESULTS In a low-endemic region, HIV-seropositive patients showed lower frequencies of PPD-specific T cells compared to immunocompetent individuals. This was not due to a general loss of immunity toward recall antigens, as T-cell immunity toward CMV or SEB was preserved. In line with continuous antigen exposure, HIV-seropositive patients from a high-endemic region showed preserved PPD-specific T-cell frequencies that were not different from those found in HIV-seronegative controls. Likewise, both groups did not differ in recall T-cell responses toward CMV or SEB. CONCLUSION A lower prevalence and frequency of PPD-specific immunity is a typical feature of HIV-related immunosuppression in low-endemic regions. In contrast, PPD-specific responses are maintained in HIV-seropositive individuals in regions with high tuberculosis prevalence. This suggests constant skewing and restriction of specific T-cell immunity toward environmental antigens in HIV-seropositive individuals.

Some key features of a mathematical description of an immune response are an estimate of the number of responding cells and the manner in which those cells divide, differentiate, and die. The intracellular dye CFSE is a powerful experimental tool for the analysis of a population of dividing cells, and numerous mathematical treatments have been aimed at using CFSE data to describe an immune response [30,31,32,37,38,42,48,49]. Recently, partial differential equation structured population models, with measured CFSE fluorescence intensity as the structure variable, have been shown to accurately fit histogram data obtained from CFSE flow cytometry experiments [18,19,52,54]. In this report, the population of cells is mathematically organized into compartments, with all cells in a single compartment having undergone the same number of divisions. A system of structured partial differential equations is derived which can be fit directly to CFSE histogram data. From such a model, cell counts (in terms of the number of divisions undergone) can be directly computed and thus key biological parameters such as population doubling time and precursor viability can be determined. Mathematical aspects of this compartmental model are discussed, and the model is fit to a data set. As in [18,19], we find temporal and division dependence in the rates of proliferation and death to be essential features of a structured population model for CFSE data. Variability in cellular autofluorescence is found to play a significant role in the data, as well. Finally, the compartmental model is compared to previous work, and statistical aspects of the experimental data are discussed.

The 1'-organyl-1,2,3,4-tetrahydrospiro[naphthalene-1,4'-piperidine] derivatives 1 a-4 a [for which organyl=benzyl (1 a), 4-methoxybenzyl (2 a), 2-phenylethyl (3 a), or 3-methylbut-2-enyl (4 a)] are high-affinity, selective σ₁ ligands. The corresponding sila-analogues 1 b-4 b (replacement of the carbon spirocenter with a silicon atom) were synthesized in multistep syntheses, starting from dichlorodivinylsilane, and were isolated as the hydrochlorides 1 b⋅HCl-4 b⋅HCl. Compounds 1 a⋅HCl-4 a⋅HCl and 1 b⋅HCl-4 b⋅HCl were structurally characterized by NMR spectroscopy (¹H, ¹³C, ²⁹Si) in solution, and the C/Si analogues 3 a⋅HCl and 3 b⋅HCl were studied by single-crystal X-ray diffraction. These structural investigations were complemented by computational studies. The σ₁ and σ₂ receptor affinities of the C/Si pairs 1 a/1 b-4 a/4 b were studied with radioligand binding assays. The σ₁ receptor affinity of the silicon compounds 1 b-4 b is slightly higher than that of the corresponding carbon analogues 1 a-4 a. Because affinity for the σ₂ receptor is decreased by the C/Si exchange, the σ₁/σ₂ selectivity of the silicon compounds is considerably improved, indicating that the C→Si switch strategy is a powerful tool for modulating both pharmacological potency and selectivity.

Advances in fluorescent labeling of cells as measured by flow cytometry have allowed for quantitative studies of proliferating populations of cells. The investigations (Luzyanina et al. in J. Math. Biol. 54:57-89, 2007; J. Math. Biol., 2009; Theor. Biol. Med. Model. 4:1-26, 2007) contain a mathematical model with fluorescence intensity as a structure variable to describe the evolution in time of proliferating cells labeled by carboxyfluorescein succinimidyl ester (CFSE). Here, this model and several extensions/modifications are discussed. Suggestions for improvements are presented and analyzed with respect to statistical significance for better agreement between model solutions and experimental data. These investigations suggest that the new decay/label loss and time dependent effective proliferation and death rates do indeed provide improved fits of the model to data. Statistical models for the observed variability/noise in the data are discussed with implications for uncertainty quantification. The resulting new cell dynamics model should prove useful in proliferation assay tracking and modeling, with numerous applications in the biomedical sciences.

The gentle and careful in vitro processing of live cells is essential in order to make them available to future therapeutic applications. We present a protocol for the activation of single-T cells based on the contact formation with individual anti-CD3/anti-CD28 presenting microbeads in a lab-on-chip environment. The chips consist of microfluidic channels and microelectrodes for performing dielectrophoretic manipulation employing a.c. electric fields. The dielectrophoretic guiding elements allow the assembly of cell-bead pairs while avoiding ill-defined physical contacts with their environment. After overnight cultivation of the manipulated cells, 77% of the bead-associated T cells expressed the activation marker molecule CD69. Physiological stress on the cells was shown to be mainly due to the single-cell cultivation and not to the manipulation in the chips. The same approach could be useful for the in vitro regulation of stem cell differentiation.

PURPOSE: Intima-media thickness (IMT) of the common carotid artery and the resistive index (RI) of the internal carotid artery correlate with the degree of atherosclerosis and are predictors of cardiovascular morbidity and mortality. Limited or no data are available about long-term predictive values and the progression of the two markers themselves. MATERIALS AND METHODS: 145 patients with at least one cardiovascular risk factor or clinically manifest atherosclerosis were included. At enrollment and after 36 and 74 months, duplex sonographic measurements of IMT CCA and RI ICA were performed. During follow-up, the occurrence of cardiovascular events (cardiovascular death, myocardial infarction, stroke) was assessed. RESULTS: At baseline, IMT was 0.79 +/- 0.16 mm and RI 0.66 +/- 0.08. Log-rank analysis showed a continuous increase in the risk of a cardiovascular event with an increasing range of IMT (p = 0.011) and RI (p = 0.006). IMT progression in patients with low versus high atherosclerotic burden (as defined by SMART score 7 points) differs significantly (32 +/- 83 microm versus 95 +/- 125 microm; p > 0.002). IMT progression was even more pronounced in patients suffering a cardiovascular event (141 +/- 105 microm versus 54 +/- 111 microm; p > 0.001). No significant RI ICA progression could be detected during follow-up in any group (patients with low vs. high atherosclerotic burden 0.00 +/- 0.06 versus 0.00 +/- 0.04; p = n. s.; patients with vs. without cardiovascular event 0.00 +/- 0.05 versus 0.01 +/- 0.03; p = n. s.). CONCLUSION: Our results confirm the predictive value for cardiovascular events of RI and IMT in long-term follow-up. In contrast to RI, IMT increases over six years, above all in patients suffering a cardiovascular event. The results suggest that IMT is suitable for cardiovascular risk prediction as well as for progression measurements, while RI cannot be recommended for progression measurements. The effect of drug therapy on RI needs furth clarification.

The positive transcription elongation factor b (P-TEFb) is a heterodimeric complex composed of cyclin-dependent kinase 9 and its regulator cyclin T1/2. It stimulates transcription elongation by phosphorylation of serine 2 residues in the carboxy-terminal domain of polymerase II. 7SK RNA and HEXIM proteins can antagonize transcriptional stimulation by sequestering P-TEFb in a catalytically inactive ribonucleoprotein (RNP). Here, we show that the previously uncharacterized La-related protein 7 (LARP7) has a role in 7SK-mediated regulation of transcription. LARP7 binds to the highly conserved 3′-terminal U-rich stretch of 7SK RNA and is an integral part of the 7SK RNP. On stimulation, LARP7 remains associated with 7SK RNA, whereas P-TEFb is released. Interestingly, reduction of LARP7 by RNA interference enhances transcription from cellular polymerase II promoters, as well as a TAT-dependent HIV-1 promoter. Thus, LARP7 is a negative transcriptional regulator of polymerase II genes, acting by means of the 7SK RNP system.

BACKGROUND: Regulatory B (Breg) and T (Treg) cells represent a biomarker for tolerance in transplant patients. Despite the importance of Treg and Breg in transplantation and the suggested crosstalk between both suppressive cell populations, little is known on how they are influenced by long-term immunosuppressive treatment. The aim of the present study was to investigate the effect of different immunosuppressive drugs used in routine clinical practice on Treg and Breg cell numbers. METHODS: Thirty-six kidney transplant recipients with stable graft function were recruited and classified according to their concomitant therapy: 22 patients received calcineurin inhibitors (CNI) and 14 patients received mammalian target of rapamycin (mTOR) inhibitors. A group of 8 healthy untreated subjects was included as control. Absolute numbers of peripheral blood-derived IL10-producing B cells (CD19(+)IL10(+)), CD19(+)CD24(hi)CD38(hi) transitional B cells and Treg cells (CD4(+)CD25(+)FOXP3(+)) were quantified in all KT patients and controls by flow cytometry. RESULTS: CD19(+)CD24(hi)CD38(hi) transitional B cells increased over time and seem to be related with long-term therapeutic graft survival irrespective of the treatment regimen. CNI and mTOR inhibitors significantly reduced numbers of Breg cells when compared with healthy individuals, whereas mTOR inhibitors expanded Treg cells in comparison with CNI drugs. CONCLUSIONS: Bridging the drug-mediated reduction of Breg cell numbers in vivo with the requirements of Breg cells for long-term transplant success remains an as yet unresolved task for therapeutic intervention. Further larger cohort studies that evaluate the effect of different treatment regimen on defined lymphocyte subpopulations are warranted. DR and MC were supported by a grant Marató TV3 137/C/2012. JP was supported by grants/nFIS-ISCIII-FEDER PI13/00598 and RedinRenRD12/0021/0024. AM was supported by a Ministerio de Economía y Competitividad grant SAF2010–21336.

A fraction of HIV-1 patients are able to generate broadly neutralizing antibodies (bNAbs) after 2 to 4 years of infection. In rare occasions such antibodies are observed close to the first year of HIV-1 infection but never within the first 6 months. In this study, we analyzed the neutralization breadth of sera from 157 antiretroviral-naive individuals who were infected for less than 1 year. A range of neutralizing activities was observed with a previously described panel of six recombinant viruses from five different subtypes (M. Medina-Ramirez et al., J Virol 85: 5804–5813, 2011, http://dx.doi.org/10.1128/JVI.02482-10 ). Some sera were broadly reactive, predominantly targeting envelope epitopes within the V2 glycan-dependent region. The neutralization breadth was positively associated with time postinfection ( P = 0.0001), but contrary to what has been reported for chronic infections, no association with the viral load was observed. Notably, five individuals within the first 6 months of infection (two as early as 77 and 96 days postinfection) showed substantial cross-neutralization. This was confirmed with an extended panel of 20 Env pseudoviruses from four different subtypes (two in tier 3, 14 in tier 2, and four in tier 1). Sera from these individuals were capable of neutralizing viruses from four different subtypes with a geometric mean 50% infective dose (ID 50 ) between 100 and 800. These results indicate that induction of cross-neutralizing responses, albeit rare, is achievable even within 6 months of HIV-1 infection. These observations encourage the search for immunogens able to elicit this kind of response in preventive HIV-1 vaccine approaches. IMPORTANCE There are very few individuals able to mount broadly neutralizing activity (bNA) close to the first year postinfection. It is not known how early in the infection cross-neutralizing responses can be induced. In the present study, we show that bNAbs, despite being rare, can be induced much earlier than previously thought. The identification of HIV-1-infected patients with these activities within the first months of infection and characterization of these responses will help in defining new immunogen designs and neutralization targets for vaccine-mediated induction of bNAbs.

Human immunodeficiency virus (HIV)-specific CD8 T-cell responses targeting products encoded within the Gag open reading frame have frequently been associated with better viral control and disease outcome during the chronic phase of HIV infection. To further clarify this relationship, we have studied the dynamics of Gag-specific CD8 T-cell responses in relation to plasma viral load and time since infection in 33 chronically infected subjects over a 9-month period. High baseline viral loads were associated with a net loss of breadth (P< 0.001) and a decrease in the total magnitude of the Gag-specific T-cell response in general (P= 0.03). Most importantly, the baseline viral load predicted the subsequent change in the breadth of Gag recognition over time (P< 0.0001,r2= 0.41). Compared to maintained responses, lost responses were low in magnitude (P< 0.0001) and subdominant in the hierarchy of Gag-specific responses. The present study indicates that chronic exposure of the human immune system to high levels of HIV viremia is a determinant of virus-specific CD8 T-cell loss.

The importance of HLA class I-restricted CD8 T-cell responses in the control of human immunodeficiency virus (HIV) infection is generally accepted. While several studies have shown an association of certain HLA class I alleles with slower disease progression, it is not fully established whether this effect is mediated by HIV-specific CD8 T-cell responses restricted by these alleles. In order to study the influence of the HLA class I alleles on the HIV-specific CD8 T-cell response and on viral control, we have assessed HIV-specific epitope recognition, plasma viral load, and expression of HLA class I alleles in a cohort of HIV-seropositive bar workers. Possession of the HLA class I alleles B5801, B8101, and B0702 was associated with a low median viral load and simultaneously with a broader median recognition of Gag epitopes compared to all other HLA alleles (twofold increase) (P= 0.0035). We further found an inverse linear relationship between the number of Gag epitopes recognized and the plasma viral load (R= −0.36;P= 0.0016). Particularly, recognition of multiple epitopes within two regions of Gag (amino acids [aa] 1 to 75 and aa 248 to 500) was associated with the maintenance of a low steady-state viremia, even years after acute infection.

West Nile virus (WNV) is an emerging arbovirus first recognized in Europe in the 1950s. Since then, outbreaks have been reported in several European countries. In 2010, the first WNV outbreak was recorded in Spain, affecting the southern part of the country. We conducted a seroprevalence study in the Catalonia region (northeastern Spain), an area considered at high risk of arbovirus transmission. A total of 800 serum samples from blood donors were collected and screened for antibodies against WNV by enzyme-linked immunosorbent assay (ELISA) and confirmed by a microneutralization assay. More than 50 samples tested positive by ELISA, but only one sample contained neutralizing antibodies against WNV and was obtained from a donor native of Pakistan. The low seroprevalence detected may serve as reference baseline data for monitoring WNV activity in our region in future years.

Understanding the fundamental steps of virus life cycles including virus–host interactions is essential for the design of effective antiviral strategies. Such understanding has been deferred by the complexity of higher eukaryotic host organisms. To circumvent experimental difficulties associated with this, systems were developed to replicate viruses in the yeast Saccharomyces cerevisiae. The systems include viruses with RNA and DNA genomes that infect plants, animals and humans. By using the powerful methodologies available for yeast genetic analysis, fundamental processes occurring during virus replication have been brought to light. Here, we review the different viruses able to direct replication and gene expression in yeast and discuss their main contributions in the understanding of virus biology.

Background and Purpose— The intima-media thickness (IMT) of the common carotid artery (CCA) is well correlated with the degree of arteriosclerosis and is a predictor of cardiovascular morbidity and mortality. The (hemodynamic) resistive index (RI) of the internal carotid artery (ICA) correlates with the degree of arteriosclerosis just as well as IMT. The aim of the study was to compare the predictive values of RI and IMT with regard to cardiovascular morbidity and mortality. Methods— A total of 146 patients with cardiovascular risk factors or established arteriosclerosis were included. Duplex sonography of the CCA and ICA was performed, and the IMT and RI were measured in both vessels. During follow-up for a median of 36 months, the occurrence of cardiovascular events (myocardial infarction, stroke, or cardiovascular death) was assessed. Results— Thirty-nine cardiovascular events occurred in 28 patients (19.2%). The relative risk for a car-diovascular event per increase of the IMT by 1 SD (0.16 mm) was 1.53 (95% CI, 1.07 to 2.18) and 1.91 (95% CI, 1.34 to 2.73) for RI ICA (0.08). The event rate in patients with IMT P =0.03) and 25.0% ( P =0.06) in patients with IMT ≥0.79 mm and RI ICA ≥0.66, respectively. Log rank analysis showed a continuous increase in the risk of cardiovascular event with increasing range of the IMT ( P =0.029) and RI ICA ( P Conclusion— The RI ICA is a predictor of cardiovascular mortality and morbidity, at least comparable to the well-established IMT.

Several prevalent and life-threatening agents enter the organism via the mucosa. In this case, a mucosal cellular immune response is essential for protection and is therefore considered the main objective of vaccination. The frequency of antigen-specific CD4+ and CD8+ T cells can be determined directly in human whole blood by a combination of surface marker and intracellular cytokine staining. Immune cells primed in the mucosal compartment also migrate through the blood and can be identified by expression of the gut-specific homing receptor alpha4beta7. Simultaneously, these lymphocytes can be functionally characterized regarding their differentiation status by analysis of CD45RO and CD27 expression and effector functions by measuring intracellular perforin or granzyme B content. Thus, the technique described in the paper is a powerful tool for clinical monitoring of the total cellular immune response to complex antigens during infection or vaccination.

It has been previously shown that the majority of human immunodeficiency virus type 1 (HIV-1)-infected splenocytes can harbour multiple, divergent proviruses with a copy number ranging from one to eight. This implies that, besides point mutations, recombination should be considered as an important mechanism in the evolution of HIV within an infected host. To explore in detail the possible contributions of multi-infection and recombination to HIV evolution, the effects of major microscopic parameters of HIV replication (i.e. the point-mutation rate, the crossover number, the recombination rate and the provirus copy number) on macroscopic characteristics (such as the Hamming distance and the abundance of n-point mutants) have been simulated in silico. Simulations predict that multiple provirus copies per infected cell and recombination act in synergy to speed up the development of sequence diversity. Point mutations can be fixed for some time without fitness selection. The time needed for the selection of multiple mutations with increased fitness is highly variable, supporting the view that stochastic processes may contribute substantially to the kinetics of HIV variation in vivo.

The gastrointestinal tract is the site of early abundant HIV replication and associated marked CD4 + T-cell depletion. The aim of this study was to characterize the basis for the increased HIV replication in this compartment. Isolated mononuclear cells of the peripheral blood (PBMCs), the intestinal lamina propria (LPMCs), and purified gut lamina propria CD4 + T-cell subpopulations (LP T cells) were isolated, phenotypically characterized, and infected in vitro with 2 different HIV-1 strains. T-cell subpopulations were analyzed by fluorescence-activated cell sorter. HIV- 1 core protein p24 was determined in supernatants after in vitro infection. Furthermore the effect of T-cell stimulation on the replication of M- and T-tropic HIV strains was studied. In vitro replication of HIV-1 was significantly increased in CD69 high compared with CD69 low CD4 + LP T cells, while there was no difference between CD103 - and CD103 + CD4 + LP T cells. Experimental stimulation of LPMCs, which mimics activation by intestinal pathogens frequently present in the bowel of HIV-infected patients, further dramatically enhances HIV replication (24.5-fold) compared with nonstimulated LPMCs. M-tropic HIV-1 showed a preferential replication in LPMCs, while T-tropic HIV-1 strain showed a preferential replication in PBMCs. Thus, the elevated activation state of target cells in the intestine and not the expression of the homing marker CD103 is directly linked to massive HIV production.

The human cellular immune response against 14 distantly related yeast species was analyzed by intracellular cytokine staining of lymphocytes after ex vivo stimulation of whole blood. While the CD4 T cell response was marginal, extensive MHC class I-restricted CD8 T cell responses were detected against a number of species including spoiling, environmental and human pathogenic yeasts. The yeast-specific CD8 T cells expressed interferon-gamma but lacked expression of CD27 and CCR7, indicating that they were end-differentiated effector memory cells. Mainly intact yeast cells rather than spheroplasts were able to induce cytokine expression in T cells demonstrating that the dominant immunogens were located in the yeast cell wall. Together these data underline the importance of the cellular immune response in protecting humans against yeast and fungal infections. And, from another perspective, recombinant yeast suggests itself as a potential vaccine candidate to efficiently induce antigen-specific CD8 T cell responses.

Blocking the PD-1/PD-L1 pathway has emerged as a potential therapy to restore impaired immune responses in human immunodeficiency virus (HIV)-infected individuals. Most reports have studied the impact of the PD-L1 blockade on effector cells and neglected possible effects on regulatory T cells (Treg cells), which play an essential role in balancing immunopathology and antiviral effector responses. The aim of this study was to define the consequences of ex vivo PD-L1 blockade on Treg cells from HIV-infected individuals. We observed that HIV infection led to an increase in PD-1+ and PD-L1+ Treg cells. This upregulation correlated with disease progression and decreased under antiretroviral treatment. Treg cells from viremic individuals had a particularly high PD-1 expression and impaired proliferative capacity in comparison with Treg cells from individuals under antiretroviral treatment. PD-L1 blockade restored the proliferative capacity of Treg cells from viremic individuals but had no effect on its suppressive capacity. Moreover, it increased the viral production in cell cultures from viremic individuals. This increase in viral production correlated with an increase in Treg cell percentage and a reduction in the CD4/Treg and CD8/Treg cell ratios. In contrast to the effect of the PD-L1 blockade on Treg cells from viremic individuals, we did not observe a significant effect on the proliferative capacity of Treg cells from individuals in whom viremia was controlled (either spontaneously or by antiretroviral treatment). However, PD-L1 blockade resulted in an increased proliferative capacity of HIV-specific-CD8 T cells in all subjects. Taken together, our findings suggest that manipulating PD-L1 in vivo can be expected to influence the net gain of effector function depending on the subject’s plasma viremia., Author Summary HIV infection causes a progressive impairment of effector immune responses, contributing to virus persistence. The restoration of these responses is essential to achieve a drug-free control over HIV. One strategy that could restore effector immune responses is the relief of the inhibitory signal displayed by the PD-1/PD-L1 pathway on effector cells. However, the PD-1/PD-L1 pathway also plays a role in the biology of regulatory T cells, which in turn suppress effector responses. Here we show that ex vivo PD-L1 blockade on peripheral blood mononuclear cells from HIV-infected individuals differentially increases the proliferative capacity of regulatory- and effector- T cells depending on the subject’s plasma viremia. Our results suggest that PD-L1 blockade will skew the effector-to-regulatory T cell ratio in favour of effector cells only in patients in whom viremia is controlled. In patients with uncontrolled viremia, PD-L1 blockade will not favour effector- T cells over regulatory- T cells, and might also boost virus reactivation. Our findings support the rationale to combine a PD-L1 blockade with antiretroviral treatment to restore effector responses in HIV-infected individuals.

Objective: Perforin is an important component of the death machinery of cytotoxic T cells (CTL). To evaluate functional differences between HIV- and cytomegalovirus (CMV)-specific CTL of coinfected patients, the frequencies of the respective perforin-expressing T cells were analysed in a rapid whole blood assay. Methods: Whole blood of HIV- and CMV-infected individuals was specifically stimulated by HIV-1 Pr55 gag or complete CMV antigen, and activation-induced intracellular cytokine and perforin expression in CD8 T cells was analysed by flow cytometry. Results: Perforin-expressing HIV-1- and CMV-specific CD8 T cells can be quantified simultaneously. Within a patient, the frequency of such HIV-specific CD8 T cells in peripheral blood was lower than the frequency of the respective CMV-specific cells. The number of the perforin-expressing HIV-specific CD8 T cells inversely correlated with the peripheral blood CD4 T cell count. Conclusions: The differential fractions of perforin-expressing virus-specific CD8 T cells in HIV and CMV double infection might be caused by differences in priming and trafficking to or from replication sites. However, without knowing the underlying mechanism, the fraction of perforin-expressing HIV-specific CD8 T cells provides another surrogate marker for disease progression.

Replication of cytomegalovirus (CMV) is largely controlled by the cellular arm of the immune response. In this study the CMV-specific CD4 T-cell response was characterized in a cohort of apparently healthy individuals. In 11% of all individuals, extremely high frequencies, between 10 and 40%, were found. High-level frequencies of CMV-specific CD4 T cells persisted over several months and were not the result of an acute infection. Specific T cells were oligoclonal and were phenotypically and functionally characterized as mature effector cells, with both cytokine-secreting and proliferative potential. These high-level frequencies do not seem to compromise the immune response towards heterologous infections, and no signs of immunopathology were observed. Whereas a large temporary expansion of virus-specific T cells is well known to occur during acute infection, we now show that extremely high frequencies of virus-specific T cells may continuously exist in chronic CMV infection without overtly compromising the remaining protective immunity.

Covering: up to April 2014 The development of drugs with broad-spectrum antiviral activities is a long pursued goal in drug discovery. It has been shown that blocking co-opted host-factors abrogates the replication of many viruses, yet the development of such host-targeting drugs has been met with scepticism mainly due to toxicity issues and poor translation to in vivo models. With the advent of new and more powerful screening assays and prediction tools, the idea of a drug that can efficiently treat a wide range of viral infections by blocking specific host functions has re-bloomed. Here we critically review the state-of-the-art in broad-spectrum antiviral drug discovery. We discuss putative targets and treatment strategies, with particular focus on natural products as promising starting points for antiviral lead development.

Although a pivotal role of proteasomes in the proteolytic generation of epitopes for major histocompatibility complex (MHC) class I presentation is undisputed, their precise function is currently the subject of an active debate: do proteasomes generate many epitopes in definitive form, or do they merely generate the COOH termini, whereas the definitive NH2 termini are cleaved by aminopeptidases? We determined five naturally processed MHC class I ligands derived from HIV-1 Nef. Unexpectedly, the five ligands correspond to only three cytotoxic T lymphocyte (CTL) epitopes, two of which occur in two COOH-terminal length variants. Parallel analyses of proteasomal digests of a Nef fragment encompassing the epitopes revealed that all five ligands are direct products of proteasomes. Moreover, in four of the five ligands, the NH2 termini correspond to major proteasome cleavage sites, and putative NH2-terminally extended precursor fragments were detected for only one of the five ligands. All ligands are transported by the transporter associated with antigen processing (TAP). The combined results from these five ligands provide strong evidence that many definitive MHC class I ligands are precisely cleaved at both ends by proteasomes. Additional evidence supporting this conclusion is discussed, along with contrasting results of others who propose a strong role for NH2-terminal trimming with direct proteasomal epitope generation being a rare event.

The semaphorin/collapsin family of proteins comprises molecules thought to be important for the guidance of growing axons. All members of this family, which includes both secreted and cell-surface molecules, share a conserved domain of approximately 500 amino acids. Here, we report the cloning of a novel murine semaphorin, termed M-sema IV. It displays 96.3% identity to the human semaphorin H-sema IV and is therefore likely to be the respective murine homologue. In addition, an isoform was identified, which contains an additional 31 amino acids in the semaphorin domain. M-sema IV appears to be expressed ubiquitously in adulthood. During embryogenesis, in situ hybridization revealed M-sema IV expression in subregions of the central nervous system and various other tissues like skin, kidney, lung and intestine.

Infections with HIV represent a great challenge for the development of strategies for an effective cure. The spectrum of diseases associated with HIV ranges from opportunistic infections and cancers to systemic physiological disorders like encephalopathy and neurocognitive impairment. A major progress in controlling HIV infection has been achieved by highly active antiretroviral therapy (HAART). However, HAART does neither eliminate the virus reservoirs in form of latently infected cells nor does it completely reconstitute immune reactivity and physiological status. Furthermore, the failure of the STEP vaccine trial and the only marginal efficacies of the RV144 trial together suggest that the causal relationships between the complex sets of viral and immunological processes that contribute to protection or disease pathogenesis are still poorly understood. Here, we provide an up-to-date overview of HIV-host interactions at the cellular, the immune system and the neuroendocrine systems level. Only by integrating this multi-level knowledge one will be able to handle the systems complexity and develop new methodologies of analysis and prediction for a functional restoration of the immune system and the health of the infected host.

HIV-specific CD8+ cytotoxic T lymphocytes (CTL) are thought to have a beneficial role in HIV infection. In a previous report we have shown that HIV-1 Nef-specific CTL can be readily induced in peripheral blood lymphocytes of seronegative healthy young adults by in vitro stimulation with autologous Epstein-Barr virus-transformed B lymphoblastoid cell lines transfected with the HIV-1 nef gene. Here we demonstrate that these Nef-specific CTL can efficiently lyse HIV-infected primary CD4+ T lymphocytes. CTL of the blood donor tested were Nef-specific and restricted by the autologous MHC class I molecules HLA-A2 and HLA-B7. They recognized HIV-1 Nef in association with both restriction elements but HIV-2 Nef only in association with HLA-B7. The cross-reactivity of the induced effector cells together with the potent immunogenicity of Nef in healthy seronegatives further support the inclusion of Nef as a constituent of HIV vaccines.

Background: Infections with HIV still represent a major human health problem worldwide and a vaccine is the only long-term option to fight efficiently against this virus. Standardized assessments of HIV-specific immune responses in vaccine trials are essential for prioritizing vaccine candidates in preclinical and clinical stages of development. With respect to neutralizing antibodies, assays with HIV-1 Env-pseudotyped viruses are a high priority. To cover the increasing demands of HIV pseudoviruses, a complete cell culture and transfection automation system has been developed. Methodology/Principal Findings: The automation system for HIV pseudovirus production comprises a modified Tecan-based Cellerity system. It covers an area of 5×3 meters and includes a robot platform, a cell counting machine, a CO2 incubator for cell cultivation and a media refrigerator. The processes for cell handling, transfection and pseudovirus production have been implemented according to manual standard operating procedures and are controlled and scheduled autonomously by the system. The system is housed in a biosafety level II cabinet that guarantees protection of personnel, environment and the product. HIV pseudovirus stocks in a scale from 140 ml to 1000 ml have been produced on the automated system. Parallel manual production of HIV pseudoviruses and comparisons (bridging assays) confirmed that the automated produced pseudoviruses were of equivalent quality as those produced manually. In addition, the automated method was fully validated according to Good Clinical Laboratory Practice (GCLP) guidelines, including the validation parameters accuracy, precision, robustness and specificity. Conclusions: An automated HIV pseudovirus production system has been successfully established. It allows the high quality production of HIV pseudoviruses under GCLP conditions. In its present form, the installed module enables the production of 1000 ml of virus-containing cell culture supernatant per week. Thus, this novel automation facilitates standardized large-scale productions of HIV pseudoviruses for ongoing and upcoming HIV vaccine trials. This article derived from a collaboration of the Fraunhofer Institute for Biomedical Engineering, the ‘‘Global HIV Vaccine Research Cryorepository Consortium’’ (principal investigator: HVB) and the ‘‘Comprehensive Antibody Vaccine Immune Monitoring Consortium’’ (principal investigator: DM) within the‘‘Collaboration for AIDS Vaccine Discovery’’, funded by the Bill and Melinda Gates Foundation (#OPP38580_01). Automation of cell culture and HIV pseudovirus production was established in exclusive cooperation with TECAN funded as a strategic investment by the Fraunhofer Society, Germany.