EBV is a B lymphotrophic gamma-herpesvirus that is associated with multiple human malignancies, including posttransplant lymphoproliferative disorder. The EBV-encoded protein, latent membrane protein 1 (LMP1), is required for oncogenic transformation of human B cells by EBV. An important consequence of LMP1 expression in EBV-infected B cells is the induction of cellular IL-10, which acts as an autocrine growth factor for B cell lymphomas. However, the mechanisms by which LMP1 induces IL-10 are incompletely understood. We previously showed that rapamycin, a clinically relevant immunosuppressant and mammalian target of rapamycin inhibitor, could suppress IL-10 production by EBV-infected B cell lines. To test the hypothesis that PI3K, which acts upstream of mammalian target of rapamycin, might also be involved in LMP1-dependent IL-10 production, we generated B cell lines expressing signaling-inducible chimeric LMP1. Our results show that induced LMP1 signaling elicits both p38- and PI3K-dependent IL-10 production in EBV- B cells. Moreover, distinct regions of the LMP1 signaling tail are associated with p38- vs PI3K-dependent IL-10 induction. We also demonstrate that the LMP1-dependent p38 and PI3K activation regulates IL-10 induction through discrete mechanisms. Whereas p38 activation is critical for the phosphorylation of the transcription factor CREB, PI3K activation is required for the inactivation of glycogen synthase kinase 3beta (GSK3beta), an inhibitory kinase that can regulate CREB function. We find that GSK3beta regulates LMP1-dependent IL-10 induction, with GSK3beta inhibition by pharmacologic or small interfering RNA strategies enhancing LMP1-induced IL-10 induction. These findings demonstrate that LMP1 uses both p38 and PI3K activation for maximal up-regulation of IL-10.

The safety and immunogenicity results from this study support inclusion of the GLA-SE adjuvant in this RSV vaccine for older adults and also support assessment of the efficacy of the vaccine in a larger clinical trial. Clinicaltrials.gov NCT02115815.

IntroductionEach B lymphocyte expresses a clonal immunoglobulin (Ig) whose heavy and light chain variable regions (V H and V L , respectively) comprise a unique collection of antigenic determinants known as the idiotype (Id). Maintenance of the Id by B cells following malignant transformation offers opportunities for the development of tumor-specific immunotherapies for B-cell malignancies. The Ig Id sequences contain epitopes that can be recognized by antibodies 1-3 and CD4 ϩ2,4-6 and CD8 ϩ T cells. 7-10 Active immunization with tumor-derived Id protein can protect mice against subsequent lymphoma challenge 2,11 and, in conjunction with chemotherapy, can cure established tumors. 12 In lymphoma patients, Id vaccination has been found to result in humoral and cellular anti-Id responses 4 that correlate with improved relapse-free and overall survival. 13 Durable tumor regressions have also been observed in several trials. 4,5,14,15 These clinical studies have relied on Id protein purified from cultures of tumormyeloma cell hybridomas, the production of which is rate-limiting for large-scale application in human trials. The ability to rapidly amplify Id-encoding DNA sequences from lymphoma cells using polymerase chain reaction (PCR) has offered the possibility of streamlining the production of custom-made Id vaccines through the application of "naked" DNA vaccination techniques. 16 Vaccination with Id-encoding DNA has proven efficacy in eliciting tumor-protective immunity in several murine lymphoma 9,[16][17][18][19] and myeloma 19 models. While this approach eliminates the need for production and purification of Id protein, repetitive immunizations are required, and immune responses are weaker than those elicited by protein vaccines, even when augmented by co-administered cytokine DNAs. 17,18 Recombinant adenoviruses offer the practical advantages of DNA vaccination in a highly efficient and immunogenic vector system. Replication-defective adenoviruses encoding an antigen of interest can be rapidly produced in high titers. 20 Adenoviruses efficiently infect a variety of target cell types in vitro or in vivo, leading to high levels of protein production in situ. Vaccination with recombinant adenoviruses has been shown to induce potent humoral [21][22][23][24][25][26][27][28] and cellular [23][24][25]27,28 immune responses to transgeneencoded products. In some cases, vaccination has resulted in breaking of immunologic tolerance to self-antigens. 27,29 In a variety of infectious disease models, adenoviral vaccination can stimulate potent, protective immunity to disease challenge. 21,22,26,28 Immunization with adenoviruses encoding model or xenogeneic tumor antigens has also been shown to induce humoral 30 and cellular [29][30][31] immune responses to the tumor antigen transgene product and to provide protective 29,30 and therapeutic 30,31 antitumor immunity.We therefore sought to determine if Id-encoding adenoviruses could induce antitumor immunity against murine lymphomas. In this report we demonstrate that in 2 dif...

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is a proven oncogene that is essential for transformation LMP14 is an EBV-encoded oncoprotein that is essential for transformation of human B lymphocytes (1-3). In B cells LMP1 mimics the CD40 receptor, although unlike CD40, LMP1 functions in a ligand-independent manner and is constitutively active (4). LMP1 activates several cellular signaling pathways culminating in expression of downstream genes involved in cell transformation, survival, and proliferation. Thus, LMP1 plays a central role in EBV-associated tumorigenesis.LMP1 is composed of a short cytoplasmic N-terminal tail required for insertion into the membrane, six membrane-spanning domains that facilitate oligomerization of the protein, and a C-terminal cytoplasmic tail. Within the C terminus of LMP1 are two major signaling domains, C-terminal activation region 1 (CTAR1) and CTAR2. The CTAR interact with tumor necrosis factor receptor-associated factors (TRAFs) and TRAF-associated death domain protein (TRADD) molecules and potentially other adapter molecules to activate NF-B, Jun N-terminal kinase (JNK), p38 mitogen-activated protein (MAP) kinase, extracellular signal-regulated kinase (Erk), and phosphoinositide 3-kinase (PI3K). Several key sites within the C terminus of LMP1 are necessary for proper signaling including the PXQXT motif in the CTAR1 region which mediates binding of TRAFs 1, 2, 3, and 5, and tyrosines 384 and 385 of CTAR2 that are essential for TRADD interaction, the recruitment of TRAF2, and association of receptor-interacting protein (5-11).Activation of NF-B by LMP1 is critical to the survival of EBV-infected B cells. NF-B activation is highly regulated through association with the IB complex that prevents nuclear localization of NF-B. In EBV-infected B cells the activation of NF-B is initiated through interaction of LMP1 with TRAF2 at CTAR1 and TRAF6 at CTAR2, allowing for activation of the IB kinase (12, 13). IB kinase phosphorylates IB␣, thereby targeting it for proteasomal degradation. NF-B is then free to translocate to the nucleus and to activate transcription of downstream genes that promotes cell survival (14,15). Similar to the NF-B pathway, interaction of the adapter protein TRAF2 with both CTAR1 and CTAR2 (indirectly through TRADD) is required for p38 MAPK activation by LMP1 (16). We (17) and others (18) have shown that activation of p38 by LMP1 contributes to the induction of IL-10, an important autocrine growth factor for EBV-infected B cells.Whereas CTAR1 and CTAR2 both contribute to signaling of NF-B and p38, the activation of JNK, PI3K/Akt, and Erk rely on a single CTAR. Specifically, the CTAR2 domain of LMP1 participates in activation of the JNK pathway through interaction with TRAF6, TRAF2, and TRADD (19 -21). JNK kinase

BackgroundIllness associated with Respiratory Syncytial Virus (RSV) remains an unmet medical need in both full-term infants and older adults. The fusion glycoprotein (F) of RSV, which plays a key role in RSV infection and is a target of neutralizing antibodies, is an attractive vaccine target for inducing RSV-specific immunity.Methodology and Principal FindingsBALB/c mice and cotton rats, two well-characterized rodent models of RSV infection, were used to evaluate the immunogenicity of intramuscularly administered RSV vaccine candidates consisting of purified soluble F (sF) protein formulated with TLR4 agonist glucopyranosyl lipid A (GLA), stable emulsion (SE), GLA-SE, or alum adjuvants. Protection from RSV challenge, serum RSV neutralizing responses, and anti-F IgG responses were induced by all of the tested adjuvanted RSV sF vaccine formulations. However, only RSV sF + GLA-SE induced robust F-specific TH1-biased humoral and cellular responses. In mice, these F-specific cellular responses include both CD4 and CD8 T cells, with F-specific polyfunctional CD8 T cells that traffic to the mouse lung following RSV challenge. This RSV sF + GLA-SE vaccine formulation can also induce robust RSV neutralizing titers and prime IFNγ-producing T cell responses in Sprague Dawley rats.Conclusions/SignificanceThese studies indicate that a protein subunit vaccine consisting of RSV sF + GLA-SE can induce robust neutralizing antibody and T cell responses to RSV, enhancing viral clearance via a TH1 immune-mediated mechanism. This vaccine may benefit older populations at risk for RSV disease.

BackgroundToll-like receptor (TLR)4 agonists are known potent immunostimulatory compounds. These compounds can be formulated as part of novel adjuvants to enhance vaccine medicated immune responses. However, the contribution of the formulation to the innate in vivo activity of TLR4 agonist compounds is not well understood.Methodology and Principal FindingsWe evaluated synthetic TLR4 agonist Glucopyranosyl Lipid A (GLA) for its effects on molecular and cellular innate immune responses in the murine model. Microarray techniques were used to compare the responses to GLA in an aqueous formulation or in an oil-in-water Stable Emulsion formulation (GLA-SE) versus either SE alone or the mineral salt aluminum hydroxide (alum) at the muscle injection site over multiple timepoints. In contrast to the minimal gene upregulation induced by SE and alum, both GLA and GLA-SE triggered MyD88- and TRIF-dependent gene expression. Genes for chemokines, cytokine receptors, signaling molecules, complement, and antigen presentation were also strongly upregulated by GLA and GLA-SE. These included chemokines for TH1-type T cells (CXCL9 and CXCL10) and mononuclear leukocytes (CCL2, CCL3) among others. GLA-SE induced stronger and more sustained gene upregulation than GLA in the muscle; GLA-SE induced genes were also detected in local draining lymph nodes and at lower levels in peripheral blood. Both GLA and GLA-SE resulted in increased cellular trafficking to the draining lymph nodes and upregulated MHC molecules and ICAM1 on local dendritic cells. GLA and GLA-SE transiently upregulated circulating MCP-1, TNFα, IFNγ and IP-10 in blood.Conclusions/SignificanceWhile GLA and GLA-SE activate a large number of shared innate genes and proteins, GLA-SE induces a quantitatively and qualitatively stronger response than GLA, SE or alum. The genes and proteins upregulated could be used to facilitate selection of appropriate adjuvant doses in vaccine formulations.

Sequential skin biopsies from six patients with severe psoriasis were studied during treatment with cyclosporin. Four of the patients cleared completely and the remaining two showed a marked improvement. A subset of dendritic cells, HLA-DR+ but lacking the T6 antigen characteristically expressed by Langerhans cells (DR+ 6-), was observed in lesional epidermis. They disappeared during treatment, before clinical improvement was apparent and at a rate which correlated with clearance of psoriasis. These cells were not found in normal or uninvolved psoriatic epidermis and their number in lesional skin appeared to be related to the clinical severity of the disease. Total numbers of CD4 and CD8, and HLA-DR+ CD8 T cells were substantially reduced in both epidermis and dermis prior to clinical improvement. In contrast, there was generally no decrease in the number of HLA-DR+ CD4 T cells in the epidermis during resolution, whereas these cells were reduced by an average of 68% in the dermis. The beneficial effects of cyclosporin in psoriasis further support the hypothesis that T cells play a central role in the pathogenesis of psoriasis. The cellular changes observed in the skin during cyclosporin treatment may help to elucidate the effects of this drug on immunoregulatory mechanisms in man.

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