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CD8(+) T cell responses restricted by MHC-E, a nonclassical MHC molecule, have been associated with protection in an SIV/rhesus macaque model. The biological relevance of HLA-E–restricted CD8(+) T cell responses in HIV infection, however, remains unknown. In this study, CD8(+) T cells responding to HIV-1 Gag peptides presented by HLA-E were analyzed. Using in vitro assays, we observed HLA-E–restricted T cell responses to what we believe to be a newly identified subdominant Gag-KL9 as well as a well-described immunodominant Gag-KF11 epitope in T cell lines derived from chronically HIV-infected patients and also primed from healthy donors. Blocking of the HLA-E/KF11 binding by the B7 signal peptide resulted in decreased CD8(+) T cell responses. KF11 presented via HLA-E in HIV-infected cells was recognized by antigen-specific CD8(+) T cells. Importantly, bulk CD8(+) T cells obtained from HIV-infected individuals recognized infected cells via HLA-E presentation. Ex vivo analyses at the epitope level showed a higher responder frequency of HLA-E–restricted responses to KF11 compared with KL9. Taken together, our findings of HLA-E–restricted HIV-specific immune responses offer intriguing and possibly paradigm-shifting insights into factors that contribute to the immunodominance of CD8(+) T cell responses in HIV infection.

// Sojan Abraham 1 , Jang-Gi Choi 1,2 , Nora M. Ortega 1 , Junli Zhang 1 , Premlata Shankar 1 and N. Manjunath Swamy 1 1 Center of Emphasis in Infectious Disease, Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX, USA 2 KM Application Center, Korea Institute of Oriental Medicine, Dong-gu, Daegu, Republic of Korea Correspondence to: N. Manjunath Swamy, email: // Keywords : interferon, HIV-1, hydrodynamic injection, Immunology and Microbiology Section, Immune response, Immunity Received : May 20, 2016 Accepted : October 02, 2016 Published : October 06, 2016 Abstract Because endogenous interferon type I (IFN-I) produced by HIV-1 infection might complicate the analysis of therapeutically administered IFN-I, we tested different humanized mouse models for induction of IFN-I during HIV-1 infection. While HIV-1 induced high levels of IFN-α in BLT mice, IFN-I was undetectable following infection in the Hu-PBL mouse model, in which only T cells expand. We therefore tested the effect of treatment with Pegylated IFN-2 (pegasys), in Hu-PBL mice. Pegasys prevented CD4 T cell depletion and reduced the viral load for 10 days, but the effect waned thereafter. We next expressed IFN-I subsets (IFN-α2, -α6, -α8, -α14, and -β) in Hu-PBL mice by hydrodynamic injection of plasmids encoding them and 2 days later infected the mice with HIV-1. CD4 T cell depletion was prevented in all subtypes of IFN-I-expressing mice by day 10. However, at day 40 post-infection, protection was seen in IFN-β- and IFN-α14-expressing mice, but not the others. The viral load followed an inverse pattern and was highest in control mice and lowest in IFN-β- and IFN-α14-expressing mice until day 40 after infection. These results show that gene therapy with plasmids encoding IFN-β and -α14, but not the commonly used -α2, confers long-term suppression of HIV-1 replication.

Hypersecretion of cytokines by innate immune cells is thought to initiate multiple organ failure in murine models of sepsis. Whether human cytokine storm also plays a similar role is not clear. Here, we show that human hematopoietic cells are required to induce sepsis-induced mortality following cecal ligation and puncture (CLP) in the severely immunodeficient nonobese diabetic (NOD)/SCID/IL2Rγ −/− mice, and siRNA treatment to inhibit HMGB1 release by human macrophages and dendritic cells dramatically reduces sepsis-induced mortality. Following CLP, compared with immunocompetent WT mice, NOD/SCID/IL2Rγ −/− mice did not show high levels of serum HMGB1 or murine proinflammatory cytokines and were relatively resistant to sepsis-induced mortality. In contrast, NOD/SCID/IL2Rγ −/− mice transplanted with human hematopoietic stem cells [humanized bone marrow liver thymic mice (BLT) mice] showed high serum levels of HMGB1, as well as multiple human but not murine proinflammatory cytokines, and died uniformly, suggesting human cytokines are sufficient to induce organ failure in this model. Moreover, targeted delivery of HMGB1 siRNA to human macrophages and dendritic cells using a short acetylcholine receptor (AchR)-binding peptide [rabies virus glycoprotein (RVG)-9R] effectively suppressed secretion of HMGB1, reduced the human cytokine storm, human lymphocyte apoptosis, and rescued humanized mice from CLP-induced mortality. siRNA treatment was also effective when started after the appearance of sepsis symptoms. These results show that CLP in humanized mice provides a model to study human sepsis, HMGB1 siRNA might provide a treatment strategy for human sepsis, and RVG-9R provides a tool to deliver siRNA to human macrophages and dendritic cells that could potentially be used to suppress a variety of human inflammatory diseases.

Flaviviruses have been shown to induce cell surface expression of major histocompatibility complex class I (MHC-I) through the activation of NF-κB. Using IKK1 −/− , IKK2 −/− , NEMO −/− , and IKK1 −/− IKK2 −/− double mutant as well as p50 −/− RelA −/− cRel −/− triple mutant mouse embryonic fibroblasts infected with Japanese encephalitis virus (JEV), we show that this flavivirus utilizes the canonical pathway to activate NF-κB in an IKK2- and NEMO-, but not IKK1-, dependent manner. NF-κB DNA binding activity induced upon virus infection was shown to be composed of RelA:p50 dimers in these fibroblasts. Type I interferon (IFN) production was significantly decreased but not completely abolished upon virus infection in cells defective in NF-κB activation. In contrast, induction of classical MHC-I (class 1a) genes and their cell surface expression remained unaffected in these NF-κB-defective cells. However, MHC-I induction was impaired in IFNAR −/− cells that lack the alpha/beta IFN receptor, indicating a dominant role of type I IFNs but not NF-κB for the induction of MHC-I molecules by Japanese encephalitis virus. Our further analysis revealed that the residual type I IFN signaling in NF-κB-deficient cells is sufficient to drive MHC-I gene expression upon virus infection in mouse embryonic fibroblasts. However, NF-κB could indirectly regulate MHC-I expression, since JEV-induced type I IFN expression was found to be critically dependent on it.

IL-10 is a crucial anti-inflammatory cytokine which can also exert a seemingly divergent immunostimulatory effects under certain conditions. We found high levels of the cytokine in a xenogeneic GVHD model where NOD-scid IL2rγcnull (NSG) mice were transplanted with human PBMCs in presence of IL-2. Presence of exogenous IL-10 altered the kinetics of IL-2 induced human T cell reconstitution in vivo, showing an initial delay, followed by rapid expansion. Further, compared to IL-2 alone, treatment with IL-2 in combination with IL-10 increased survival in most animals and completely protected ∼20% of mice from GVHD. Additionally, IL-2 induced expansion of both CD4+ and CD8+ xenoreactive T cells whereas a combination of IL-2 and IL-10 resulted in selective expansion of CD4+ T cells only. TCR Vβ repertoire analysis of CD4+ T cells showed that in contrast to IL-2 alone, simultaneous presence of both cytokines drastically reduced the Vβ repertoire of the expanded CD4+ T cells. Highly restricted Vβ usage was also observed when the cytokine combination was tested in an allogeneic GVHD model where NOD-scid IL2rγcnull mice expressing HLA-DR4 (NSG-DR4) were transplanted with purified CD4+ T cells from HLA-DR4 negative donors. Taken together, our results demonstrate that IL-10 can profoundly modulate the subset composition and repertoire of responding T cells during GVHD.

Although patients with GVHD have elevated serum levels of IL10, whether its role is protective or pathogenic remains unclear. Here, we used a humanized mouse model to study the role of IL-10 in GVHD. When human PBMCs were engrafted in NOD-scid IL2rγc(null) mice expressing human IL-10, the T cells underwent massive expansion resulting in lethality by day 21, whereas control mice survived for at least 40 days. Histopathology of the liver showed extensive mononuclear cell infiltration in IL-10 expressing but not in control mice. Corresponding to their aggressiveness, the T cells in the IL-10 group exhibited predominantly an effector memory phenotype (CD45RO(+)CD27(-)) while in control mice, the T cells were of transitional memory phenotype (CD45RO(+)CD27(+)). Further, IL-10 receptor blocking antibody was able to protect the animals from GVHD. Since our results demonstrate a direct pathogenic role for IL-10, blockade of IL-10 signaling may provide a therapeutic option for GVHD.

Targeting DNA vaccines to dendritic cells (DCs) greatly enhances immunity. Although several approaches have been used to target protein antigens to DCs, currently there is no method that targets DNA vaccines directly to DCs. Here, we show that a small peptide derived from the rabies virus glycoprotein, fused to protamine residues (RVG-P) can target DNA to myeloid cells, including DCs, that results in enhanced humoral and T-cell responses. DCs targeted with a DNA vaccine encoding the immunodominant vaccinia B8R gene via RVG-P were able to restimulate vaccinia-specific memory T cells in vitro. Importantly, a single i.v. injection of B8R gene bound to RVG-P was able prime a vaccinia-specific T-cell response that was able to rapidly clear a subsequent vaccinia challenge in mice. Moreover, delivery of DNA in DCs was enough to induce DC maturation and efficient antigen presentation without the need for adjuvants. Finally, immunization of mice with a DNA-vaccine encoding West Nile virus (WNV) prM and E proteins via RVG-P elicited high titers of WN neutralizing antibodies that protected mice from lethal WNV challenge. Thus, RVG-P provides a reagent to target DNA vaccines to myeloid cells and elicit robust T-cell and humoral immune responses.

Butyroyloxymethyl diethylphosphate (AN-7) is a prodrug of butyric acid effective in reducing cardiotoxicity caused by chemotherapy. In this study, we tested whether AN-7 protects the heart and cardiomyocytes against ischemia injury. A single oral dose of AN-7 was given to mice or rats. Animals were sacrificed 1.5 or 24 h later and the hearts were subjected to ischemia and reperfusion ex-vivo (Langendorff). The mechanical performance was recorded throughout and the infarct size was measured at the end of reperfusion. Neonatal rat cardiomyocytes were subjected to 24-48 h hypoxia (1% O(2)) in the absence or presence of AN-7 and mitochondria damage and cell death were assessed. Proteins were analyzed by Western immunoblotting. In the two rodents, a single dose of AN-7 given in vivo preconditioned the hearts for improved functional recovery from ischemia and reperfusion performed ex-vivo. Both 1.5 h and 24 h treatments improved the pressure-related parameters whereas the coronary flow was ameliorated in the 24 h treatment only. Infarct size was smaller in the AN-7 treated hearts. In cardiomyocytes, AN-7 diminished the hypoxia induced dissipation of mitochondria membrane potential and cell death. Compared with untreated controls, AN-7-treated hearts recovering from global ischemia and cardiomyocytes undergoing hypoxia, displayed significantly higher levels of the cytoprotective heme oxygenase-1. Our findings indicate that AN-7 imparts cardioprotection against ischemia both in vivo and in vitro and emerges as a potential treatment modality for cardiac injury.

Japanese encephalitis virus (JEV) is a flavivirus that is one of the major causes of encephalitis in Eastern and Southern Asia with recent observations marking its spread to newer geographical regions. Flaviviruses, in general consitutute a global health threat since many of them are endemic in large parts of the Americas, Africa and Australia in addition to Asia and suitable vaccines are unavailable for many of them. JEV causes serious inflammation of the brain, which may lead to permanent brain damage, and has a high mortality rate. More than three billion people live in JE endemic areas and JEV is estimated to cause 45,000 human cases of disease and 10000 deaths per year (Solomon and Winter, 2004; van den Hurk et al., 2009). The occurrence of encephalitic epidemics in humans due to this virus have been recognized from 1870s in Japan where it was termed Japanese B encephalitis to distinguish it from type A encephalitis caused by sleeping sickness. The first prototype Nakayama strain was isolated and established in 1935 from the brain of a fatal case from Tokyo, Japan. This was closely followed by virus isolation from Culex tritaeniorrhynchus in 1936 confirming earlier suspicions that mosquitoes served as the transmission vector (Solomon et al., 2000). JEV transmission occurs through a zoonotic cycle involving mosquitoes as an important intermediate maintenance and replicative vector. Vertebrates, chiefly pigs and ardeid birds act as amplifying hosts. Only those animals that develop high viraemias are significant in this natural enzootic cycle. Humans are infected only coincidently when bitten by an infected mosquito and are dead end hosts. Human to human transmission is yet to be documented, possibly due to the presence of transient viraemia. The absence of high serum viraemia prevents the virus from being picked up during mosquito bites. Pigs play a major role in the transmission cycle with respect to human infection since they are often bred close to humans whereas herons, egrets and other ardeid birds are important for maintenance and overwintering of this virus. Among the other vertebrates, horses also develop central nervous system (CNS) infections and are considered as dead end hosts. Rodents are relatively refractory to infections while amphibians, reptiles and bats can also be infected (Mackenzie et al., 2004). JEV does not cause encephalitis in pigs and birds although it is

Dengue is a common arthropod-borne flaviviral infection in the tropics, for which there is no vaccine or specific antiviral drug. The infection is often associated with serious complications such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), in which both viral and host factors have been implicated. RNA interference (RNAi) is a potent antiviral strategy and a potential therapeutic option for dengue if a feasible strategy can be developed for delivery of small interfering RNA (siRNA) to dendritic cells (DCs) and macrophages, the major in vivo targets of the virus and also the source of proinflammatory cytokines. Here we show that a dendritic cell-targeting 12-mer peptide (DC3) fused to nona- d -arginine (9dR) residues (DC3-9dR) delivers siRNA and knocks down endogenous gene expression in heterogenous DC subsets, (monocyte-derived DCs [MDDCs], CD34 + hematopoietic stem cell [HSC])-derived Langerhans DCs, and peripheral blood DCs). Moreover, DC3-9dR-mediated delivery of siRNA targeting a highly conserved sequence in the dengue virus envelope gene (siFvE D ) effectively suppressed dengue virus replication in MDDCs and macrophages. In addition, DC-specific delivery of siRNA targeting the acute-phase cytokine tumor necrosis factor alpha (TNF-α), which plays a major role in dengue pathogenesis, either alone or in combination with an antiviral siRNA, significantly reduced virus-induced production of the cytokine in MDDCs. Finally to validate the strategy in vivo , we tested the ability of the peptide to target human DCs in the NOD/SCID/IL-2Rγ −/− mouse model engrafted with human CD34 + hematopoietic stem cells (HuHSC mice). Treatment of mice by intravenous (i.v.) injection of DC3-9dR-complexed siRNA targeting TNF-α effectively suppressed poly(I:C)-induced TNF-α production by DCs. Thus, DC3-9dR can deliver siRNA to DCs both in vitro and in vivo , and this delivery approach holds promise as a therapeutic strategy to simultaneously suppress virus replication and curb virus-induced detrimental host immune responses in dengue infection.

Nonclassical MHC Class 1b antigens differ from classical MHC class 1a antigens in having a restricted polymorphism as well as varied surface expression in different cell types. They have been hypothesized to play a role in bridging adaptive and innate immune responses.We examined the effects of JEV infection on the expression of classical MHC class 1a and nonclassical MHC class 1b genes in five different cell lines. Among the nonclassical genes, H-2Q4 was induced in H-6 hepatoma, primary astrocytes, mouse embryo fibroblasts, L929 and 3T3 cells. H-2T23 and H-2T10 genes were not induced in H-6 and 3T3, respectively, but were induced in the other cell lines examined. Both H-2Q4 encoded Qb1 and H-2T23 encoded Qa-$1^b$ antigens were induced on the cell surface upon JEV infection in primary astrocytes and mouse embryonic fibroblasts. Classical MHC-I genes and the genes associated with antigen presentation such as Tap1, Tap2, Tapasin, Lmp2, Lmp7 and Lmp10 as well as type 1 (\alpha/\beta) IFNs were induced in all cell lines. However, IFN\gamma was not induced. Further, induction of H-2Q4 and H-2T23 by JEV was independent of NF-\kappa B but type 1 IFN dependent while H-2T10 was dependent on NF-\kappa B and type 1 IFN independent. Thus, while classical MHC genes were induced by JEV in all cell lines tested despite high levels of constitutive expression in L929 and 3T3, nonclassical genes were not inducible in all cell lines tested and involved different mechanisms of induction.

Infection with Flaviviruses upregulates the cell surface expression of MHC-I,MHC-II, ICAM- I (CD54),VCAM- I (CD106) and TAP proteins. Although all these studies have been confirmed using West Nile virus and other Flaviviruses, there are few reports that have examined the effects of Japanese encephalitis virus (JEV) infection directly on nonclassical and classical MHC expression in astrocytes. We show in this report that JEV infection of mouse brain astrocytes results in induction of the nonclassical MHC Class Ib genes, H-2T23, H-2Q4 and H-2T10 in addition to MHC-I, Type I (alpha,beta) IFNs, TAP-1, TAP-2, Tapasin, LMP-2, LMP-7 and LMP-10 but not IFN gamma, CD80, CD86 and MHC-II genes. The increased cel I surface expression of these antigens as well as induction of the genes mentioned above as measured by RT-PCR suggests that JEV infection may lead to the induction of classical MHC Class la as well as nonclassical MHC Class lb molecules.