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The molecular contamination of an animal facility was investigated during and after an infection with highly pathogenic African swine fever virus (ASFV) among domestic pigs. The investigation evaluated the risk of indirect transmission of the disease and indicated points that may facilitate cleaning and disinfection processes.

Immunotherapy with checkpoint inhibitors, albeit commonly used against tumors, is still at its infancy against chronic virus infections. It relies on the reinvigoration of exhausted T lymphocytes to eliminate virus-infected cells. Since T cell exhaustion is a physiological process to reduce immunopathology, the reinvigoration of these cells might be associated with an augmentation of pathological changes. To test this possibility, we here analyzed in the model system of chronic lymphocytic choriomeningitis virus (LCMV)-infected mice whether treatment with the checkpoint inhibitor anti-PD-L1 antibody would increase CD8 T cell-dependent fibrosis. We show that pre-existing spleen fibrosis did not worsen under conditions that increase CD8 T cell functionality and reduce virus loads suggesting that the CD8 T cell functionality increase remained below its pathogenicity threshold. These promising findings should further encourage immunotherapeutic trials against chronic virus infections.

African swine fever (ASF) is a deadly disease of swine currently causing a worldwide pandemic, leading to severe economic consequences for the porcine industry. The control of disease spread is hampered by the limitation of available effective vaccines. Live attenuated vaccines (LAVs) are currently the most advanced vaccine prototypes, providing strong protection against ASF. However, the significant advances achieved using LAVs must be complemented with further studies to analyze vaccine-induced immunity. Here, we characterized the onset of cross-protective immunity triggered by the LAV candidate BA71ΔCD2. Intranasally vaccinated pigs were challenged with the virulent Georgia 2007/1 strain at days 3, 7 and 12 postvaccination. Only the animals vaccinated 12 days before the challenge had effectively controlled infection progression, showing low virus loads, minor clinical signs and a lack of the unbalanced inflammatory response characteristic of severe disease. Contrarily, the animals vaccinated 3 or 7 days before the challenge just showed a minor delay in disease progression. An analysis of the humoral response and whole blood transcriptome signatures demonstrated that the control of infection was associated with the presence of virus-specific IgG and a cytotoxic response before the challenge. These results contribute to our understanding of protective immunity induced by LAV-based vaccines, encouraging their use in emergency responses in ASF-affected areas.

Developing prolonged antigen delivery systems that mimic long-term exposure to pathogens appears as a promising but still poorly explored approach to reach durable immunities. In this study, we have used a simple technology by which His-tagged proteins can be assembled, assisted by divalent cations, as supramolecular complexes with progressive complexity, namely protein-only nanoparticles and microparticles. Microparticles produced out of nanoparticles are biomimetics of secretory granules from the mammalian hormonal system. Upon subcutaneous administration, they slowly disintegrate, acting as an endocrine-like secretory system and rendering the building block nanoparticles progressively bioavailable. The performance of such materials, previously validated for drug delivery in oncology, has been tested here regarding the potential for time-prolonged antigen release. This has been completed by taking, as a building block, a nanostructured version of p30, a main structural immunogen from the African swine fever virus (ASFV). By challenging the system in both mice and pigs, we have observed unusually potent pro-inflammatory activity in porcine macrophages, and long-lasting humoral and cellular responses in vivo, which might overcome the need for an adjuvant. The robustness of both innate and adaptive responses tag, for the first time, these dynamic depot materials as a novel and valuable instrument with transversal applicability in immune stimulation and vaccinology.

Acute infection and chronic infection are the two most common fates of pathogenic virus infections. While several factors that contribute to these fates are described, the critical control points and the mechanisms that underlie infection fate regulation are incompletely understood. Using the acute and chronic lymphocytic choriomeningitis virus (LCMV) infection model of mice, we find that the early dynamic pattern of the IFN-I response is a differentiating trait between both infection fates. Acute-infected mice generate a 2-wave IFN-I response while chronic-infected mice generate only a 1-wave response. The underlying cause is a temporal difference in CD8 T cell-mediated killing of splenic marginal zone CD169+ macrophages. It occurs later in acute infection and thus enables CD169+ marginal zone macrophages to produce the 2nd IFN-I wave. This is required for subsequent immune events including induction of inflammatory macrophages, generation of effector CD8+ T cells and virus clearance. Importantly, these benefits come at a cost for the host in the form of spleen fibrosis. Due to an earlier marginal zone destruction, these ordered immune events are deregulated in chronic infection. Our findings demonstrate the critical importance of kinetically well-coordinated sequential immune events for acute infection control and highlights that it may come at a cost for the host organism., (© 2023. The Author(s).)

Latency is a major barrier towards virus elimination in HIV-1-infected individuals. Yet, the mechanisms that contribute to the maintenance of HIV-1 latency are incompletely understood. Here we describe the Schlafen 12 protein (SLFN12) as an HIV-1 restriction factor that establishes a post-transcriptional block in HIV-1-infected cells and thereby inhibits HIV-1 replication and virus reactivation from latently infected cells. The inhibitory activity is dependent on the HIV-1 codon usage and on the SLFN12 RNase active sites. Within HIV-1-infected individuals, SLFN12 expression in PBMCs correlated with HIV-1 plasma viral loads and proviral loads suggesting a link with the general activation of the immune system. Using an RNA FISH-Flow HIV-1 reactivation assay, we demonstrate that SLFN12 expression is enriched in infected cells positive for HIV-1 transcripts but negative for HIV-1 proteins. Thus, codon-usage dependent translation inhibition of HIV-1 proteins participates in HIV-1 latency and can restrict the amount of virus release after latency reversal., (© 2023. The Author(s).)

This study aimed to evaluate the effects of feeding spray-dried porcine plasma (SDPP) on the protection afforded by the BA71∆CD2 African swine fever virus (ASFV) vaccine prototype. Two groups of pigs acclimated to diets without or with 8% SDPP were intranasally inoculated with 10 5 plaque-forming units (PFU) of live attenuated ASFV strain BA71∆CD2 and, three weeks later, left in direct contact with pigs infected with the pandemic Georgia 2007/01 ASFV strain. During the post-exposure (pe) period, 2/6 from the conventional diet group showed a transient peak rectal temperature >40.5 °C before day 20 pe, and some tissue samples collected at 20 d pe from 5/6 were PCR+ for ASFV, albeit showing Ct values much higher than Trojan pigs. Interestingly, the SDPP group did not show fever, neither PCR+ in blood nor rectal swab at any time pe, and none of the postmortem collected tissue samples were PCR+ for ASFV. Differential serum cytokine profiles among groups at vaccination, and a higher number of ASFV-specific IFNϒ-secreting T cells in pigs fed with SDPP soon after the Georgia 2007/01 encounter, confirmed the relevance of Th1-like responses in ASF protection. We believe that our result shows that nutritional interventions might contribute to improving future ASF vaccination strategies.

The contribution of cross-presenting XCR1+ dendritic cells (DCs) and SIRPα+ DCs in maintaining T cell function during exhaustion and immunotherapeutic interventions of chronic infections remains poorly characterized. Using the mouse model of chronic LCMV infection, we found that XCR1+ DCs are more resistant to infection and highly activated compared with SIRPα+ DCs. Exploiting XCR1+ DCs via Flt3L-mediated expansion or XCR1-targeted vaccination notably reinvigorates CD8+ T cells and improves virus control. Upon PD-L1 blockade, XCR1+ DCs are not required for the proliferative burst of progenitor exhausted CD8+ T (T PEX ) cells but are indispensable to sustain the functionality of exhausted CD8+ T (T EX ) cells. Combining anti-PD-L1 therapy with increased frequency of XCR1+ DCs improves functionality of T PEX and T EX subsets, while increase of SIRPα+ DCs dampened their proliferation. Together, this demonstrates that XCR1+ DCs are crucial for the success of checkpoint inhibitor-based therapies through differential activation of exhausted CD8+ T cell subsets., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

African swine fever virus (ASFV) is causing a worldwide pandemic affecting the porcine industry and leading to important global economic consequences. The virus causes a highly lethal hemorrhagic disease in wild boars and domestic pigs. Lack of effective vaccines hampers the control of virus spread, thus increasing the pressure on the scientific community for urgent solutions. However, knowledge on the immune components associated with protection is very limited. Here we characterized the in vitro recall response induced by immune cells from pigs intranasally vaccinated with the BA71ΔCD2 deletion mutant virus. Vaccination conferred dose-dependent cross-protection associated with both ASFV-specific antibodies and IFNγ-secreting cells. Importantly, bulk and single-cell transcriptomics of blood and lymph node cells from vaccinated pigs revealed a positive feedback from adaptive to innate immunity. Indeed, activation of Th1 and cytotoxic T cells was concomitant with a rapid IFNγ-dependent triggering of an inflammatory response characterized by TNF-producing macrophages, as well as CXCL10-expressing lymphocytes and cross-presenting dendritic cells. Altogether, this study provides a detailed phenotypic characterization of the immune cell subsets involved in cross-protection against ASFV, and highlights key functional immune mechanisms to be considered for the development of an effective ASF vaccine., Competing Interests: The authors declare no competing interests., (Copyright: © 2022 Bosch-Camós et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Highly pathogenic avian influenza viruses (HPAIVs) cause severe systemic disease and high mortality rates in chickens, leading to a huge economic impact in the poultry sector. However, some chickens are resistant to the disease. This study aimed at evaluating the mechanisms behind HPAIV disease resistance. Chickens of different breeds were challenged with H7N1 HPAIV or clade 2.3.4.4b H5N8 HPAIV, euthanized at 3 days post-inoculation (dpi), and classified as resistant or susceptible depending on the following criteria: chickens that presented i) clinical signs, ii) histopathological lesions, and iii) presence of HPAIV antigen in tissues were classified as susceptible, while chickens lacking all these criteria were classified as resistant. Once classified, we performed RNA-Seq from lung and spleen samples in order to compare the transcriptomic signatures between resistant and susceptible chickens. We identified minor transcriptomic changes in resistant chickens in contrast with huge alterations observed in susceptible chickens. Interestingly, six differentially expressed genes were downregulated in resistant birds and upregulated in susceptible birds. Some of these genes belong to the NF-kappa B and/or mitogen-activated protein kinase signaling pathways. Among these six genes, the serine protease-encoding gene PLAU was of particular interest, being the most significantly downregulated gene in resistant chickens. Expression levels of this protease were further validated by RT-qPCR in a larger number of experimentally infected chickens. Furthermore, HPAIV quasi-species populations were constructed using 3 dpi oral swabs. No substantial changes were found in the viral segments that interact with the innate immune response and with the host cell receptors, reinforcing the role of the immune system of the host in the clinical outcome. Altogether, our results suggest that an early inactivation of important host genes could prevent an exaggerated immune response and/or viral replication, conferring resistance to HPAIV in chickens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Perlas, Argilaguet, Bertran, Sánchez-González, Nofrarías, Valle, Ramis, Cortey and Majó.)

A fundamental unsolved issue in vaccine design is how neutralizing antibodies and cytotoxic CD8 + T cells cooperate numerically in controlling virus infections. We hypothesize on a viewpoint for the multiplicative cooperativity between neutralizing antibodies and CD8 + T cells and propose how this might be exploited for improving vaccine-induced protective immunity., Competing Interests: Declaration of interests The authors have nothing to declare., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

African swine fever (ASF) has become the major threat to the global swine industry. Lack of available commercial vaccines complicates the implementation of global control strategies. So far, only live attenuated ASF viruses (ASFV) have demonstrated solid protection efficacy at the experimental level. The implementation of molecular techniques has allowed the generation of a collection of deletion mutants lacking ASFV-specific virulence factors, some of them with promising potential as vaccine candidates against the pandemic genotype II ASFV strain currently circulating in Africa, Europe, Asia and Oceania. Despite promising results, there is room for improvement, mainly from the biosafety point of view. Aiming to improve the safety of BA71∆CD2, a cross-protective recombinant live attenuated virus (LAV) lacking the ASFV CD2v gene (encoding β-glucuronidase as a reporter gene) available in our laboratory, three new recombinants were generated using BA71∆CD2 as a template: the single mutant BA71∆CD2 f , this time containing the fluorescent mCherry reporter gene instead of CD2v, and two double recombinants lacking CD2v and either the lectin gene (EP153R) or the uridine kinase (UK) gene (DP96R). Comparative in vivo experiments using BA71∆CD2 f , BA71∆CD2DP96R and BA71∆CD2EP153R recombinant viruses as immunogens, demonstrated that deletion of either DP96R or EP153R from BA71∆CD2 f decreases vaccine efficacy and does not improve safety. Our results additionally confirm ASFV challenge as the only available method today to evaluate the protective efficacy of any experimental vaccine. We believe that understanding the fine equilibrium between attenuation and inducing protection in vivo deserves further study and might contribute to more rational vaccine designs in the future.

African swine fever (ASF) has become the major threat for the global swine industry. Furthermore, the epidemiological situation of African swine fever virus (ASFV) in some endemic regions of Sub-Saharan Africa is worse than ever, with multiple virus strains and genotypes currently circulating in a given area. Despite the recent advances on ASF vaccine development, there are no commercial vaccines yet, and most of the promising vaccine prototypes available today have been specifically designed to fight the genotype II strains currently circulating in Europe, Asia, and Oceania. Previous results from our laboratory have demonstrated the ability of BA71∆CD2, a recombinant LAV lacking CD2v, to confer protection against homologous (BA71) and heterologous genotype I (E75) and genotype II (Georgia2007/01) ASFV strains, both belonging to same clade (clade C). Here, we extend these results using BA71∆CD2 as a tool trying to understand ASFV cross-protection, using phylogenetically distant ASFV strains. We first observed that five out of six (83.3%) of the pigs immunized once with 10 6 PFU of BA71∆CD2 survived the tick-bite challenge using Ornithodoros sp. soft ticks naturally infected with RSA/11/2017 strain (genotype XIX, clade D). Second, only two out of six (33.3%) survived the challenge with Ken06.Bus (genotype IX, clade A), which is phylogenetically more distant to BA71∆CD2 than the RSA/11/2017 strain. On the other hand, homologous prime-boosting with BA71∆CD2 only improved the survival rate to 50% after Ken06.Bus challenge, all suffering mild ASF-compatible clinical signs, while 100% of the pigs immunized with BA71∆CD2 and boosted with the parental BA71 virulent strain survived the lethal challenge with Ken06.Bus, without almost no clinical signs of the disease. Our results confirm that cross-protection is a multifactorial phenomenon that not only depends on sequence similarity. We believe that understanding this complex phenomenon will be useful for designing future vaccines for ASF-endemic areas.

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.

Type I interferon (IFN-I) provides effective antiviral immunity but can exacerbate harmful inflammatory reactions and cause hematopoietic stem cell (HSC) exhaustion; therefore, IFN-I expression must be tightly controlled. While signaling mechanisms that limit IFN-I induction and function have been extensively studied, less is known about transcriptional repressors acting directly on IFN-I regulatory regions. We show that NFAT5, an activator of macrophage pro-inflammatory responses, represses Toll-like receptor 3 and virus-induced expression of IFN-I in macrophages and dendritic cells. Mice lacking NFAT5 exhibit increased IFN-I production and better control of viral burden upon LCMV infection but show exacerbated HSC activation under systemic poly(I:C)-induced inflammation. We identify IFNβ as a primary target repressed by NFAT5, which opposes the master IFN-I inducer IRF3 by binding to an evolutionarily conserved sequence in the IFNB1 enhanceosome that overlaps a key IRF site. These findings illustrate how IFN-I responses are balanced by simultaneously opposing transcription factors., (© 2019 Huerga Encabo et al.)

The novel therapies with immune checkpoint inhibitors hold great promises for patients with chronic virus infections and cancers. This is based mainly on the partial reversal of the exhausted phenotype of antigen-specific cytotoxic CD8 T cells (CTL). Recently, we have shown that the restoration of HIV-specific T cell function depends on the HIV infection stage of an infected individual. Here we aimed to answer two fundamental questions: (i) Can one estimate growth parameters for the HIV-specific proliferative responsiveness upon PD-L1 blockade ex vivo? (ii) Can one use these parameter estimates to predict clinical benefit for HIV-infected individuals displaying diverse infection phenotypes? To answer these questions, we first analyzed HIV-1 Gag-specific CD8 T cell proliferation by time-resolved CFSE assays and estimated the effect of PD-L1 blockade on division and death rates, and specific precursor frequencies. These values were then incorporated into a model for CTL-mediated HIV control and the effects on CTL frequencies, viral loads and CD4 T cell counts were predicted for different infection phenotypes. The biggest absolute increase in CD4 T cell counts was in the group of slow progressors while the strongest reduction in virus loads was observed in progressor patients. These results suggest a significant clinical benefit only for a subgroup of HIV-infected individuals. However, as PD1 is a marker of lymphocyte activation and expressed on several lymphocyte subsets including also CD4 T cells and B cells, we subsequently examined the multiple effects of anti-PD-L1 blockade beyond those on CD8 T cells. This extended model then predicts that the net effect on HIV load and CD4 T cell number depends on the interplay between positive and negative effects of lymphocyte subset activation. For a physiologically relevant range of affected model parameters, PD-L1 blockade is likely to be overall beneficial for HIV-infected individuals., Competing Interests: The authors have declared that no competing interests exist.

The processes and mechanisms of virus infection fate decisions that are the result of a dynamic virus-immune system interaction with either an efficient effector response and virus elimination or an alleviated immune response and chronic infection are poorly understood. Here, we characterized the host response to acute and chronic lymphocytic choriomeningitis virus (LCMV) infections by gene coexpression network analysis of time-resolved splenic transcriptomes. First, we found an early attenuation of inflammatory monocyte/macrophage prior to the onset of T cell exhaustion, and second, a critical role of the XCL1-XCR1 communication axis during the functional adaptation of the T cell response to the chronic infection state. These findings not only reveal an important feedback mechanism that couples T cell exhaustion with the maintenance of a lower level of effector T cell response but also suggest therapy options to better control virus levels during the chronic infection phase., (© 2019 Argilaguet et al.; Published by Cold Spring Harbor Laboratory Press.)

The host immune response against infection requires the coordinated action of many diverse cell subsets that dynamically adapt to a pathogen threat. Due to the complexity of such a response, most immunological studies have focused on a few genes, proteins, or cell types. With the development of "omic"-technologies and computational analysis methods, attempts to analyze and understand complex system dynamics are now feasible. However, the decomposition of transcriptomic data sets generated from complete organs remains a major challenge. Here, we combined Weighted Gene Coexpression Network Analysis (WGCNA) and Digital Cell Quantifier (DCQ) to analyze time-resolved mouse splenic transcriptomes in acute and chronic Lymphocytic Choriomeningitis Virus (LCMV) infections. This enabled us to generate hypotheses about complex immune functioning after a virus-induced perturbation. This strategy was validated by successfully predicting several known immune phenomena, such as effector cytotoxic T lymphocyte (CTL) expansion and exhaustion. Furthermore, we predicted and subsequently verified experimentally macrophage-CD8 T cell cooperativity and the participation of virus-specific CD8 + T cells with an early effector transcriptome profile in the host adaptation to chronic infection. Thus, the linking of gene expression changes with immune cell kinetics provides novel insights into the complex immune processes within infected tissues.

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., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

African swine fever virus (ASFV) is the causal agent of African swine fever, a hemorrhagic and often lethal porcine disease causing enormous economical losses in affected countries. Endemic for decades in most of the sub-Saharan countries and Sardinia, the risk of ASFV-endemicity in Europe has increased since its last introduction into Europe in 2007. Live attenuated viruses have been demonstrated to induce very efficient protective immune responses, albeit most of the time protection was circumscribed to homologous ASFV challenges. However, their use in the field is still far from a reality, mainly due to safety concerns. In this study we compared the course of the in vivo infection caused by two homologous ASFV strains: the virulent E75 and the cell cultured adapted strain E75CV1, obtained from adapting E75 to grow in the CV1 cell-line. Interestingly, the kinetics of both viruses not only differed on the clinical signs that they caused and in the virus loads found, but also in the immunological pathways activated throughout the infections. Furthermore, E75CV1 confirmed its protective potential against the homologous E75 virus challenge and allowed the demonstration of poor cross-protection against BA71, thus defining it as heterologous. The in vitro specificity of the CD8(+) T-cells present at the time of lethal challenge showed a clear activation against the homologous virus (E75) but not against BA71. These findings will be of utility for a better understanding of ASFV pathogenesis and for the rational designing of safe and efficient vaccines against this virus.

Virus infections represent complex biological systems governed by multiple-level regulatory processes of virus replication and host immune responses. Understanding of the infection means an ability to predict the systems behaviour under various conditions. Such predictions can only rely upon quantitative mathematical models. The model formulations should be tightly linked to a fundamental step called "coordinatization" (Hermann Weyl), that is, the definition of observables, parameters, and structures that enable the link with a biological phenotype. In this review, we analyse the mathematical modelling approaches to LCMV infection in mice that resulted in quantification of some fundamental parameters of the CTL-mediated virus control including the rates of T cell turnover, infected target cell elimination, and precursor frequencies. We show how the modelling approaches can be implemented to address diverse aspects of immune system functioning under normal conditions and in response to LCMV and, importantly, make quantitative predictions of the outcomes of immune system perturbations. This may highlight the notion that data-driven applications of meaningful mathematical models in infection biology remain a challenge.

Unlabelled: African swine fever is one of the most devastating pig diseases, against which there is no vaccine available. Recent work from our laboratory has demonstrated the protective potential of DNA vaccines encoding three African swine fever viral antigens (p54, p30, and the hemagglutinin extracellular domain) fused to ubiquitin. Partial protection was afforded in the absence of detectable antibodies prior to virus challenge, and survival correlated with the presence of a large number of hemagglutinin-specific CD8(+) T cells in blood. Aiming to demonstrate the presence of additional CD8(+) T-cell determinants with protective potential, an expression library containing more than 4,000 individual plasmid clones was constructed, each one randomly containing a Sau3AI restriction fragment of the viral genome (p54, p30, and hemagglutinin open reading frames [ORFs] excluded) fused to ubiquitin. Immunization of farm pigs with the expression library yielded 60% protection against lethal challenge with the virulent E75 strain. These results were further confirmed by using specific-pathogen-free pigs after challenging them with 10(4) hemadsorbing units (HAU) of the cell culture-adapted strain E75CV1. On this occasion, 50% of the vaccinated pigs survived the lethal challenge, and 2 out of the 8 immunized pigs showed no viremia or viral excretion at any time postinfection. In all cases, protection was afforded in the absence of detectable specific antibodies prior to challenge and correlated with the detection of specific T-cell responses at the time of sacrifice. In summary, our results clearly demonstrate the presence of additional protective determinants within the African swine fever virus (ASFV) genome and open up the possibility for their future identification., Importance: African swine fever is a highly contagious disease of domestic and wild pigs that is endemic in many sub-Saharan countries, where it causes important economic losses and is currently in continuous expansion across Europe. Unfortunately, there is no treatment nor an available vaccine. Early attempts using attenuated vaccines demonstrated their potential to protect pigs against experimental infection. However, their use in the field remains controversial due to safety issues. Although inactive and subunit vaccines did not confer solid protection against experimental ASFV infection, our DNA vaccination results have generated new expectations, confirming the key role of T-cell responses in protection and the existence of multiple ASFV antigens with protective potential, more of which are currently being identified. Thus, the future might bring complex and safe formulations containing more than a single viral determinant to obtain broadly protective vaccines. We believe that obtaining the optimal vaccine formulation it is just a matter of time, investment, and willingness., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

A recently developed class of models incorporating the cyton model of population generation structure into a conservation-based model of intracellular label dynamics is reviewed. Statistical aspects of the data collection process are quantified and incorporated into a parameter estimation scheme. This scheme is then applied to experimental data for PHA-stimulated CD4+T and CD8+T cells collected from two healthy donors. This novel mathematical and statistical framework is shown to form the basis for accurate, meaningful analysis of cellular behaviour for a population of cells labelled with the dye carboxyfluorescein succinimidyl ester and stimulated to divide.

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.

CFSE analysis of a proliferating cell population is a popular tool for the study of cell division and divisionlinked changes in cell behavior. Recently Banks et al. (2011), Luzyanina et al. (2009), Luzyanina et al. (2007), a partial differential equation (PDE) model to describe lymphocyte dynamics in a CFSE proliferation assay was proposed. We present a significant revision of this model which improves the physiological understanding of several parameters. Namely, the parameter used previously as a heuristic explanation for the dilution of CFSE dye by cell division is replaced with a more physical component, cellular autofluorescence. The rate at which label decays is also quantified using a Gompertz decay process. We then demonstrate a revised method of fitting the model to the commonly used histogram representation of the data. It is shown that these improvements result in a model with a strong physiological basis which is fully capable of replicating the behavior observed in the data., (Copyright © 2011 Elsevier B.V. All rights reserved.)

One of the main criticisms to DNA vaccines is the poor immunogenicity that they confer on occasions, at least in large animals. Confirming this theory, immunization with plasmid DNA encoding two African swine fever virus genes in frame (pCMV-PQ), failed in inducing detectable immune responses in pigs, while it was successful in mice. Aiming to improve the immune responses induced in swine, a new plasmid was constructed, encoding the viral genes fused in frame with a single chain variable fragment of an antibody specific for a swine leukocyte antigen II (pCMV-APCH1PQ). Our results clearly demonstrate that targeting antigens to antigen professional cells exponentially enhanced the immune response induced in pigs, albeit that the DNA vaccine was not able to confer protection against lethal viral challenge. Indeed, a viremia exacerbation was observed in each of the pigs that received the pCMV-APCH1PQ plasmid, this correlating with the presence of non-neutralizing antibodies and antigen-specific SLA II-restricted T-cells. The implications of our discoveries for the development of future vaccines against African swine fever virus and other swine pathogens are discussed., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In this study, a new in situ hybridisation (ISH) protocol has been developed to identify African swine fever virus (ASFV) genome in formalin-fixed, paraffin-embedded tissues. Different digoxigenin labelled ASFV-probes were tested, including single ASFV-specific oligonucleotides, an 18.5kb restriction fragment from the viral genome and the entire ASFV genome. The latter showed the highest sensitivity in all tissues tested, independently of the virus used for challenge: E75L or Ba71L. Although a similar ASFV genome distribution was observed, the number of ISH-positive cells was higher for Ba71L compared to E75L infected tissues. As expected, the monocyte-macrophage cell lineage was the main target cell for ASFV infection. Corresponding with the last stages of infection, ISH-positive signals were also found in other cell types, including endothelial cells, hepatocytes and neutrophils. Furthermore, two unexpected findings were also noticed: the detection of a specific ISH-signal in lymphocytes and a tendency to find the signal in the nucleus of infected cells. In summary, the present findings demonstrate the utility of this new ISH protocol to study ASFV pathogenesis and its potential use as a diagnostic tool., (Copyright 2010 Elsevier B.V. All rights reserved.)

Classical swine fever (CSF) is a highly contagious viral infection affecting domestic and wild pigs. For classical swine fever virus (CSFV), immunization with plasmids expressing different versions of glycoprotein E2 has proven an effective way to induce protection. Previously, we have also shown that immunization with DNA vaccine expressing glycoprotein E2 (DNA-E2) induced specific T helper cell responses in the absence of neutralizing antibodies. However, the role of T cell responses in protection against CSFV is largely unknown. Here we have extended these studies to deeply characterize the role of T cell responses by a DNA-E2 and their correlation with protection against CSFV infection. Thus, pigs vaccinated with the DNA vaccine induced a strong cellular immune response, characterized by the specific induction IFN-gamma expressing T cells after vaccination without any detectable levels of CSFV neutralizing antibodies. Constant levels of CSFV-specific IFN-gamma producing cells observed from the beginning of the infection until 7 days after challenge in vaccinated animals might contribute to early control of CSFV replication, at least until neutralizing antibodies are developed. Severe clinical signs of the disease, including high titers of viremia, pyrexia and virus spread to different organs, were recorded in the non-vaccinated challenged animals, in comparison to the vaccinated animals where only one animal showed mild clinical signs and a short peak of viremia. Lack of complete protection in this animal correlated with a delay on the induction of neutralizing antibodies, detectable only from day 11 post-CSFV challenge. Conversely, the rest of the pigs within the group developed neutralizing antibodies as early as at day two post-challenge, correlating with sterile protection. Finally, an inverse correlation seemed to exist between early induction of IFN-alpha and the protection observed, while IL-10 seemed to be differentially regulated in vaccinated and non-vaccinated animals. Our results support the relevance of the induction of a strong T cellular response to confer a solid protection upon DNA vaccination against CSFV. Further experiments are needed to be done in order to clarify the key cytokines playing a role in CSFV-protection and to obtain emergency vaccines capable to confer robust and fast protection., (Copyright 2009 Elsevier B.V. All rights reserved.)