Viral Sequestration of Antigen Subverts Cross Presentation to CD8+ T Cells

Virus-specific CD8+ T cells (TCD8+) are initially triggered by peptide-MHC Class I complexes on the surface of professional antigen presenting cells (pAPC). Peptide-MHC complexes are produced by two spatially distinct pathways during virus infection. Endogenous antigens synthesized within virus-infected pAPC are presented via the direct-presentation pathway. Many viruses have developed strategies to subvert direct presentation. When direct presentation is blocked, the cross-presentation pathway, in which antigen is transferred from virus-infected cells to uninfected pAPC, is thought to compensate and allow the generation of effector TCD8+. Direct presentation of vaccinia virus (VACV) antigens driven by late promoters does not occur, as an abortive infection of pAPC prevents production of these late antigens. This lack of direct presentation results in a greatly diminished or ablated TCD8+ response to late antigens. We demonstrate that late poxvirus antigens do not enter the cross-presentation pathway, even when identical antigens driven by early promoters access this pathway efficiently. The mechanism mediating this novel means of viral modulation of antigen presentation involves the sequestration of late antigens within virus factories. Early antigens and cellular antigens are cross-presented from virus-infected cells, as are late antigens that are targeted to compartments outside of the virus factories. This virus-mediated blockade specifically targets the cross-presentation pathway, since late antigen that is not cross-presented efficiently enters the MHC Class II presentation pathway. These data are the first to describe an evasion mechanism employed by pathogens to prevent entry into the cross-presentation pathway. In the absence of direct presentation, this evasion mechanism leads to a complete ablation of the TCD8+ response and a potential replicative advantage for the virus. Such mechanisms of viral modulation of antigen presentation must also be taken into account during the rational design of antiviral vaccines

Novel Role for the AnxA1-Fpr2/ALX Signaling Axis as a Key Regulator of Platelet Function to Promote Resolution of Inflammation

Background: Ischemia reperfusion injury (I/RI) is a common complication of cardiovascular diseases. Resolution of detrimental I/RI-generated prothrombotic and proinflammatory responses is essential to restore homeostasis. Platelets play a crucial part in the integration of thrombosis and inflammation. Their role as participants in the resolution of thromboinflammation is underappreciated; therefore we used pharmacological and genetic approaches, coupled with murine and clinical samples, to uncover key concepts underlying this role. Methods: Middle cerebral artery occlusion with reperfusion was performed in wild-type or annexin A1 (AnxA1) knockout (AnxA1-/-) mice. Fluorescence intravital microscopy was used to visualize cellular trafficking and to monitor light/dye-induced thrombosis. The mice were treated with vehicle, AnxA1 (3.3 mg/kg), WRW4 (1.8 mg/kg), or all 3, and the effect of AnxA1 was determined in vivo and in vitro. Results: Intravital microscopy revealed heightened platelet adherence and aggregate formation post I/RI, which were further exacerbated in AnxA1-/- mice. AnxA1 administration regulated platelet function directly (eg, via reducing thromboxane B2 and modulating phosphatidylserine expression) to promote cerebral protection post-I/RI and act as an effective preventative strategy for stroke by reducing platelet activation, aggregate formation, and cerebral thrombosis, a prerequisite for ischemic stroke. To translate these findings into a clinical setting, we show that AnxA1 plasma levels are reduced in human and murine stroke and that AnxA1 is able to act on human platelets, suppressing classic thrombin-induced inside-out signaling events (eg, Akt activation, intracellular calcium release, and Ras-associated protein 1 [Rap1] expression) to decrease IIbβ3 activation without altering its surface expression. AnxA1 also selectively modifies cell surface determinants (eg, phosphatidylserine) to promote platelet phagocytosis by neutrophils, thereby driving active resolution. (n=5-13 mice/group or 7-10 humans/group.) Conclusions: AnxA1 affords protection by altering the platelet phenotype in cerebral I/RI from propathogenic to regulatory and reducing the propensity for platelets to aggregate and cause thrombosis by affecting integrin (IIbβ3) activation, a previously unknown phenomenon. Thus, our data reveal a novel multifaceted role for AnxA1 to act both as a therapeutic and a prophylactic drug via its ability to promote endogenous proresolving, antithromboinflammatory circuits in cerebral I/RI. Collectively, these results further advance our knowledge and understanding in the field of platelet and resolution biology.Fil: Senchenkova, Elena Y.. State University of Louisiana; Estados UnidosFil: Ansari, Junaid. State University of Louisiana; Estados UnidosFil: Becker, Felix. University Hospital Muenster; AlemaniaFil: Vital, Shantel A.. State University of Louisiana; Estados UnidosFil: Al-Yafeai, Zaki. State University of Louisiana; Estados UnidosFil: Sparkenbaugh, Erica M.. University North Carolina Chapel Hill; Estados UnidosFil: Pawlinski, Rafal. University North Carolina Chapel Hill; Estados UnidosFil: Stokes, Karen Y.. State University of Louisiana; Estados UnidosFil: Carroll, Jennifer L.. State University of Louisiana; Estados UnidosFil: Dragoi, Ana-Maria. State University of Louisiana; Estados UnidosFil: Qin, Cheng Xue. Baker Heart And Diabetes Institute; AustraliaFil: Ritchie, Rebecca H.. Baker Heart And Diabetes Institute; AustraliaFil: Sun, Hai. University Hospital Muenster; AlemaniaFil: Cuellar-Saenz, Hugo H.. State University of Louisiana; Estados UnidosFil: Rubinstein Guichon, Mara Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina. Columbia University; Estados UnidosFil: Han, Yiping W.. Columbia University; Estados UnidosFil: Orr, A. Wayne. University Hospital Muenster; AlemaniaFil: Perretti, Mauro. Queen Mary University Of London; Reino UnidoFil: Granger, D. Neil. State University of Louisiana; Estados UnidosFil: Gavins, Felicity N.E.. State University of Louisiana; Estados Unido

A framework for human microbiome research

A variety of microbial communities and their genes (the microbiome) exist throughout the human body, with fundamental roles in human health and disease. The National Institutes of Health (NIH)-funded Human Microbiome Project Consortium has established a population-scale framework to develop metagenomic protocols, resulting in a broad range of quality-controlled resources and data including standardized methods for creating, processing and interpreting distinct types of high-throughput metagenomic data available to the scientific community. Here we present resources from a population of 242 healthy adults sampled at 15 or 18 body sites up to three times, which have generated 5,177 microbial taxonomic profiles from 16S ribosomal RNA genes and over 3.5 terabases of metagenomic sequence so far. In parallel, approximately 800 reference strains isolated from the human body have been sequenced. Collectively, these data represent the largest resource describing the abundance and variety of the human microbiome, while providing a framework for current and future studies

Structure, function and diversity of the healthy human microbiome

Author Posting. © The Authors, 2012. This article is posted here by permission of Nature Publishing Group. The definitive version was published in Nature 486 (2012): 207-214, doi:10.1038/nature11234.Studies of the human microbiome have revealed that even healthy individuals differ remarkably in the microbes that occupy habitats such as the gut, skin and vagina. Much of this diversity remains unexplained, although diet, environment, host genetics and early microbial exposure have all been implicated. Accordingly, to characterize the ecology of human-associated microbial communities, the Human Microbiome Project has analysed the largest cohort and set of distinct, clinically relevant body habitats so far. We found the diversity and abundance of each habitat’s signature microbes to vary widely even among healthy subjects, with strong niche specialization both within and among individuals. The project encountered an estimated 81–99% of the genera, enzyme families and community configurations occupied by the healthy Western microbiome. Metagenomic carriage of metabolic pathways was stable among individuals despite variation in community structure, and ethnic/racial background proved to be one of the strongest associations of both pathways and microbes with clinical metadata. These results thus delineate the range of structural and functional configurations normal in the microbial communities of a healthy population, enabling future characterization of the epidemiology, ecology and translational applications of the human microbiome.This research was supported in part by National Institutes of Health grants U54HG004969 to B.W.B.; U54HG003273 to R.A.G.; U54HG004973 to R.A.G., S.K.H. and J.F.P.; U54HG003067 to E.S.Lander; U54AI084844 to K.E.N.; N01AI30071 to R.L.Strausberg; U54HG004968 to G.M.W.; U01HG004866 to O.R.W.; U54HG003079 to R.K.W.; R01HG005969 to C.H.; R01HG004872 to R.K.; R01HG004885 to M.P.; R01HG005975 to P.D.S.; R01HG004908 to Y.Y.; R01HG004900 to M.K.Cho and P. Sankar; R01HG005171 to D.E.H.; R01HG004853 to A.L.M.; R01HG004856 to R.R.; R01HG004877 to R.R.S. and R.F.; R01HG005172 to P. Spicer.; R01HG004857 to M.P.; R01HG004906 to T.M.S.; R21HG005811 to E.A.V.; M.J.B. was supported by UH2AR057506; G.A.B. was supported by UH2AI083263 and UH3AI083263 (G.A.B., C. N. Cornelissen, L. K. Eaves and J. F. Strauss); S.M.H. was supported by UH3DK083993 (V. B. Young, E. B. Chang, F. Meyer, T. M. S., M. L. Sogin, J. M. Tiedje); K.P.R. was supported by UH2DK083990 (J. V.); J.A.S. and H.H.K. were supported by UH2AR057504 and UH3AR057504 (J.A.S.); DP2OD001500 to K.M.A.; N01HG62088 to the Coriell Institute for Medical Research; U01DE016937 to F.E.D.; S.K.H. was supported by RC1DE0202098 and R01DE021574 (S.K.H. and H. Li); J.I. was supported by R21CA139193 (J.I. and D. S. Michaud); K.P.L. was supported by P30DE020751 (D. J. Smith); Army Research Office grant W911NF-11-1-0473 to C.H.; National Science Foundation grants NSF DBI-1053486 to C.H. and NSF IIS-0812111 to M.P.; The Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231 for P.S. C.; LANL Laboratory-Directed Research and Development grant 20100034DR and the US Defense Threat Reduction Agency grants B104153I and B084531I to P.S.C.; Research Foundation - Flanders (FWO) grant to K.F. and J.Raes; R.K. is an HHMI Early Career Scientist; Gordon&BettyMoore Foundation funding and institutional funding fromthe J. David Gladstone Institutes to K.S.P.; A.M.S. was supported by fellowships provided by the Rackham Graduate School and the NIH Molecular Mechanisms in Microbial Pathogenesis Training Grant T32AI007528; a Crohn’s and Colitis Foundation of Canada Grant in Aid of Research to E.A.V.; 2010 IBM Faculty Award to K.C.W.; analysis of the HMPdata was performed using National Energy Research Scientific Computing resources, the BluBioU Computational Resource at Rice University

Dendritic Cell Migration Limits the Duration of CD8+ T-Cell Priming to Peripheral Viral Antigen▿

CD8+ T cells (TCD8+) play a crucial role in immunity to viruses. Antiviral TCD8+ are initially activated by recognition of major histocompatibility complex (MHC) class I-peptide complexes on the surface of professional antigen-presenting cells (pAPC). Migration of pAPC from the site of infection to secondary lymphoid organs is likely required during a natural infection. Migrating pAPC can be directly infected with virus or may internalize antigen derived from virus-infected cells. The use of experimental virus infections to assess the requirement for pAPC migration in initiation of TCD8+ responses has proven difficult to interpret because injected virus can readily drain to secondary lymphoid organs without the need for cell-mediated transport. To overcome this ambiguity, we examined the generation of antigen-specific TCD8+ after immunization with recombinant adenoviruses that express antigen driven by skin-specific or ubiquitous promoters. We show that the induction of TCD8+ in response to tissue-targeted antigen is less efficient than the response to ubiquitously expressed antigen and that the resulting TCD8+ fail to clear all target cells pulsed with the antigenic peptide. This failure to prime a fully functional TCD8+ response results from a reduced period of priming to peripherally expressed antigen versus ubiquitously expressed antigen and correlated with a brief burst of pAPC migration from the skin, a requirement for induction of the response to peripheral antigen. These results indicate that a reduced duration of pAPC migration after virus infection likely reduces the amplitude of the TCD8+ response, allowing persistence of the peripheral virus

Improved Immune Responses in Young and Aged Mice with Adjuvanted Vaccines against H1N1 Influenza Infection

Elderly people are at high risk for influenza-related morbidity and mortality due to progressive immunosenescence. While toll-like receptor (TLR) agonist containing adjuvants, and other adjuvants, have been shown to enhance influenza vaccine-induced protective responses, the mechanisms underlying how these adjuvanted vaccines could benefit the elderly remain elusive. Here, we show that a split H1N1 influenza vaccine (sH1N1) combined with a TLR4 agonist, glucopyranosyl lipid adjuvant formulated in a stable oil-in-water emulsion (GLA-SE), boosts IgG2c:IgG1 ratios, enhances hemagglutination inhibition (HAI) titers, and increases protection in aged mice. We find that all adjuvanted sH1N1 vaccines tested were able to protect both young and aged mice from lethal A/H1N1/California/4/2009 virus challenge after two immunizations compared to vaccine alone. We show that GLA-SE combined with sH1N1, however, also provides enhanced protection from morbidity in aged mice given one immunization (based on change in weight percentage). While the GLA-SE-adjuvanted sH1N1 vaccine promotes the generation of cytokine-producing T helper 1 cells, germinal center B cells, and long-lived bone marrow plasma cells in young mice, these responses were muted in aged mice. Differential in vitro responses, dependent on age, were also observed from mouse-derived bone marrow-derived dendritic cells and lung homogenates following stimulation with adjuvants, including GLA-SE. Besides enhanced HAI titers, additional protective factors elicited with sH1N1 + GLA-SE in young mice were observed, including (a) rapid reduction of viral titers in the lung, (b) prevention of excessive lung inflammation, and (c) homeostatic maintenance of alveolar macrophages (AMs) following H1N1 infection. Collectively, our results provide insight into mechanisms of adjuvant-mediated immune protection in the young and elderly

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<p>Elderly people are at high risk for influenza-related morbidity and mortality due to progressive immunosenescence. While toll-like receptor (TLR) agonist containing adjuvants, and other adjuvants, have been shown to enhance influenza vaccine-induced protective responses, the mechanisms underlying how these adjuvanted vaccines could benefit the elderly remain elusive. Here, we show that a split H1N1 influenza vaccine (sH1N1) combined with a TLR4 agonist, glucopyranosyl lipid adjuvant formulated in a stable oil-in-water emulsion (GLA-SE), boosts IgG2c:IgG1 ratios, enhances hemagglutination inhibition (HAI) titers, and increases protection in aged mice. We find that all adjuvanted sH1N1 vaccines tested were able to protect both young and aged mice from lethal A/H1N1/California/4/2009 virus challenge after two immunizations compared to vaccine alone. We show that GLA-SE combined with sH1N1, however, also provides enhanced protection from morbidity in aged mice given one immunization (based on change in weight percentage). While the GLA-SE-adjuvanted sH1N1 vaccine promotes the generation of cytokine-producing T helper 1 cells, germinal center B cells, and long-lived bone marrow plasma cells in young mice, these responses were muted in aged mice. Differential in vitro responses, dependent on age, were also observed from mouse-derived bone marrow-derived dendritic cells and lung homogenates following stimulation with adjuvants, including GLA-SE. Besides enhanced HAI titers, additional protective factors elicited with sH1N1 + GLA-SE in young mice were observed, including (a) rapid reduction of viral titers in the lung, (b) prevention of excessive lung inflammation, and (c) homeostatic maintenance of alveolar macrophages (AMs) following H1N1 infection. Collectively, our results provide insight into mechanisms of adjuvant-mediated immune protection in the young and elderly.</p

image_1.PDF

<p>Elderly people are at high risk for influenza-related morbidity and mortality due to progressive immunosenescence. While toll-like receptor (TLR) agonist containing adjuvants, and other adjuvants, have been shown to enhance influenza vaccine-induced protective responses, the mechanisms underlying how these adjuvanted vaccines could benefit the elderly remain elusive. Here, we show that a split H1N1 influenza vaccine (sH1N1) combined with a TLR4 agonist, glucopyranosyl lipid adjuvant formulated in a stable oil-in-water emulsion (GLA-SE), boosts IgG2c:IgG1 ratios, enhances hemagglutination inhibition (HAI) titers, and increases protection in aged mice. We find that all adjuvanted sH1N1 vaccines tested were able to protect both young and aged mice from lethal A/H1N1/California/4/2009 virus challenge after two immunizations compared to vaccine alone. We show that GLA-SE combined with sH1N1, however, also provides enhanced protection from morbidity in aged mice given one immunization (based on change in weight percentage). While the GLA-SE-adjuvanted sH1N1 vaccine promotes the generation of cytokine-producing T helper 1 cells, germinal center B cells, and long-lived bone marrow plasma cells in young mice, these responses were muted in aged mice. Differential in vitro responses, dependent on age, were also observed from mouse-derived bone marrow-derived dendritic cells and lung homogenates following stimulation with adjuvants, including GLA-SE. Besides enhanced HAI titers, additional protective factors elicited with sH1N1 + GLA-SE in young mice were observed, including (a) rapid reduction of viral titers in the lung, (b) prevention of excessive lung inflammation, and (c) homeostatic maintenance of alveolar macrophages (AMs) following H1N1 infection. Collectively, our results provide insight into mechanisms of adjuvant-mediated immune protection in the young and elderly.</p