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Standard assays based on ELISA and RT-PCR have been widely used to detect flaviviral infections, including the Zika virus (Zika). Despite their simple, unique, and sensitive features, RT-PCR and ELISA-based assays cannot meet the requirements of high-throughput screening of bulk samples during an outbreak. Several research groups around the world are working on the development of rapid, multiplex, and sensitive assays to overcome the limitations of standard assays used in viral detection. Recent advances in flow cytometry have led to remarkable progress in its use as a basic analysis tool in laboratories. Here, we used the advantages of flow cytometry to develop a Zika detection assay using recombinant Zika envelope (E) protein. The E protein-based flow cytometry assay was able to detect anti-Zika E antibodies from Zika-infected patients, Zika-infected mice, and mice immunized with recombinant Zika E protein. We report the development of the first flow cytometry-based diagnostic assay that can be used for Zika detection. Its rapid turnaround time and ability to detect antibodies from Zika-infected patients can be used to improve the diagnostic accuracy of Zika detection. Keywords: Flavivirus; Zika virus; E protein; NS-1 protein; flow cytometry; ELISA; RT-PCR.

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.

A DNA vaccine encoding prM and E protein has been shown to induce protection against Zika virus (ZIKV) infection in mice and monkeys. However, its effectiveness in humans remains undefined. Moreover, identification of which immune cell types are specifically infected in humans is unclear. We show that human myeloid cells and B cells are primary targets of ZIKV in humanized mice. We also show that a DNA vaccine encoding full length prM and E protein protects humanized mice from ZIKV infection. Following administration of the DNA vaccine, humanized DRAG mice developed antibodies targeting ZIKV as measured by ELISA and neutralization assays. Moreover, following ZIKV challenge, vaccinated animals presented virtually no detectable virus in human cells and in serum, whereas unvaccinated animals displayed robust infection, as measured by qRT-PCR. Our results utilizing humanized mice show potential efficacy for a targeted DNA vaccine against ZIKV in humans., Highlights • Zika DNA vaccine elicits protective antibody response in humanized DRAG mice. • Human myeloid cells and B cells are targets of Zika virus. A Zika virus vaccine requires testing in human immune cells to determine its effectiveness. In this communication, we report that a DNA vaccine encoding Zika prM and E protein is able to elicit protective neutralizing antibodies against Zika virus in humanized DRAG mice, and that human myeloid cells and B cells are primary targets of Zika virus.

The aim of the present work was to prepare and evaluate sublingual fast dissolving niosomal films containing Nifedipine with the purpose to achieve quick onset of action. Niosomes are utilized for prolonged release of the drug, and its film increases bioavailability via the sublingual route. Niosomes were prepared using span 60 and cholesterol at different drug to surfactant ratios. The niosomes were characterized for particle size and zeta-potential. The selected niosomal formulation was incorporated into polymeric films using hydroxypropyl methyl cellulose E15 and methyl cellulose as film-forming polymers and crosspovidone, croscarmelose sodium and sodium starch glycollite as superdisintegrants to produce six formulations. The physical characteristics of the prepared films were studied, in addition to evaluating the in vitro drug release and stability. The formulation F6 containing the methyl cellulose and sodium starch glycolate as good film forming polymer and superdisintegrant respectively was found to be the best formulation showing good film forming capacity, tensile strength and % cumulative drug release of 93.91±0.648% after 9 minutes. The kinetic data analysis showed that it follows zero order models by non fickian release mechanism. These results indicate that the fast dissolving niosomal film could be a promising delivery system to enhance the bioavailability and prolong the therapeutic effect of Nifedipine.

Ranitidine hydrochloride (BCS class III) is mainly used for the treatment of gastric ulcer and esophagititis, but its low bioavailability leads to shorter half-life. The aim of this research work is to formulate ranitidine floating micro sponges to improve the gastric residence time and sustained release action. Ranitidine floating micro sponges were prepared by quasi emulsion solvent diffusion method using the polymers ethyl cellulose, eudragit S and eudragit RS and their combination to produce six formulations (F1 – F6). FTIR reports confirmed the absence of incompatibilities between the drug and excipients. This formulation was evaluated for micromeritic properties, particle size, encapsulation efficiency, percent buoyancy and percentage cumulative drug release. The formulation F6 containing the combination of ethyl cellulose and eudragit RS was found to be the best formulation showing 86 ± 1.7% buoyancy, 62.58±0.71 % encapsulation efficiency and % cumulative drug release of 94.50±0.07% after 8 hrs. The kinetic data analysis showed that it follows zero order model by non fickian release mechanism. Therefore, this ranitidine floating micro sponges provides a promising novel approach for the treatment of gastric ulcer.

Multiplexed miRNA-based shRNAs (shRNA-miRs) could have wide potential to simultaneously suppress multiple genes. Here, we describe a simple strategy to express a large number of shRNA-miRs using minimal flanking sequences from multiple endogenous miRNAs. We found that a sequence of 30 nucleotides flanking the miRNA duplex was sufficient for efficient processing of shRNA-miRs. We inserted multiple shRNAs in tandem, each containing minimal flanking sequence from a different miRNA. Deep sequencing of transfected cells showed accurate processing of individual shRNA-miRs and that their expression did not decrease with the distance from the promoter. Moreover, each shRNA was as functionally competent as its singly expressed counterpart. We used this system to express one shRNA-miR targeting CCR5 and six shRNA-miRs targeting the HIV-1 genome. The lentiviral construct was pseudotyped with HIV-1 envelope to allow transduction of both resting and activated primary CD4 T cells. Unlike one shRNA-miR, the seven shRNA-miR transduced T cells nearly abrogated HIV-1 infection in vitro. Additionally, when PBMCs from HIV-1 seropositive individuals were transduced and transplanted into NOD/SCID/IL-2R γc(-/-) mice (Hu-PBL model) efficient suppression of endogenous HIV-1 replication with restoration of CD4 T cell counts was observed. Thus, our multiplexed shRNA appears to provide a promising gene therapeutic approach for HIV-1 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.

The CRISPR/Cas9 system provides an easy way to edit specific site/s in the genome and thus offers tremendous opportunity for human gene therapy for a wide range of diseases. However, one major concern is off-target effects, particularly with long-term expression of Cas9 nuclease when traditional expression methods such as via plasmid/viral vectors are used. To overcome this limitation, we pre-packaged Cas9 protein (Cas9P LV) in lentiviral particles for transient exposure and showed its effectiveness for gene disruption in cells, including primary T cells expressing specific single guide RNAs (sgRNAs). We then constructed an 'all in one virus' to express sgRNAs in association with pre-packaged Cas9 protein (sgRNA/Cas9P LV). We successfully edited CCR5 in TZM-bl cells by this approach. Using an sgRNA-targeting HIV long terminal repeat, we also were able to disrupt HIV provirus in the J-LAT model of viral latency. Moreover, we also found that pre-packaging Cas9 protein in LV particle reduced off-target editing of chromosome 4:-29134166 locus by CCR5 sgRNA, compared with continued expression from the vector. These results show that sgRNA/Cas9P LV can be used as a safer approach for human gene therapy applications.

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.

CCR5 disruption by zinc finger nucleases (ZFNs) is a promising method for HIV-1 gene therapy. However, successful clinical translation of this strategy necessitates the development of a safe and effective method for delivery into relevant cells. We used non-integrating lentivirus (NILV) for transient expression of ZFNs and pseudotyped the virus with HIV-envelope for targeted delivery to CD4(+) T cells. Both activated and resting primary CD4(+) T cells transduced with CCR5-ZFNs NILV showed resistance to HIV-1 infection in vitro. Furthermore, NILV transduced resting CD4(+) T cells from HIV-1 seronegative individuals were resistant to HIV-1 challenge when reconstituted into NOD-scid IL2rγc null (NSG) mice. Likewise, endogenous virus replication was suppressed in NSG mice reconstituted with CCR5-ZFN-transduced resting CD4(+) T cells from treatment naïve as well as ART-treated HIV-1 seropositive patients. Taken together, NILV pseudotyped with HIV envelope provides a simple and clinically viable strategy for HIV-1 gene therapy.

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.

HIV and SIV disease progression is associated with chronic expression of type I IFN and IFN-stimulated genes. A recent study showed that a high-dose injection of IFN-1 early during primary SIV infection in the rhesus macaque model was protective, while neutralization of endogenous IFN-1 was deleterious. In contrast, in the same animal model, prolonged IFN-1 administration accelerated disease development in the chronic stage of the infection. To better understand the role of IFN-1 during HIV infection, we used humanized-PBL mouse model for this study. The advantage of this model is that there is no endogenous interferon production upon HIV infection, allowing analysis of IFN-1 therapy. Since continuous presence of IFN-1 is detrimental in the SIV model, we hypothesized that intermittent treatment with IFN-1 may be protective against HIV infection. To test this hypothesis, we carried out intermittent pegasys (peg-interferon alpha) treatment after HIV infection in human PBL infused NOD-scid IL2rγcnull (hu-PBL) mice. This treatment with pegasys diminished HIV replication and delayed CD4 depletion compared to control mice. To further test the role of IFN, we are currently using humanized BLT mouse model of HIV infection. HIV infection in BLT mice leads to continuous production of high levels of type-I IFNs; and thus, we are studying the effects of continuous or intermittent IFN blockade using type I IFN antagonist. The results will be discussed.

Dendritic cell targeting of antigens greatly enhances immunogenicity. Although there are several reagents (DEC205, Dectin-1, Langerin, Clec9, nanoparticle, etc) to target protein antigens to DCs, there is still no method to target 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 DCs resulting in enhanced T-cell and humoral responses. Targeted delivery of DNA vaccine encoding the immunodominant Vaccinia virus B8R gene to DCs with RVG-P was able to re-stimulate Vaccinia-specific memory T cells in vitro. Moreover, a single iv injection of B8R gene bound to RVG-P without any adjuvants was able prime a Vaccinia-specific T-cell response that was able to rapidly clear a subsequent Vaccinia virus challenge in mice. Finally, immunization of mice with a DNA vaccine encoding with 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 tool to target DNA vaccines to DCs to elicit robust T-cell and humoral immune responses.

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.

High level of IL-10 is present in sera of patients with Graft-versus-host disease (GVHD) following allogenic bone marrow transplantation. Our studies with NSG mouse model of GVHD induced by transfer of human PBMCs also revealed high levels of IL-10 in the sera, particularly in presence of IL-2, which accelerates the disease. Blocking antibody to IL-10 was unable to protect the animals from GVHD progression. To directly test the effect of IL-10, GVHD was induced after hydrodynamic injection of plasmids encoding human IL-10 singly or in combination with IL-2. When administered alone, IL-10 treatment resulted in more severe GVHD manifestation. Further, in IL-2 accelerated GVHD, IL-10 was unable to ameliorate the course of the disease. There was a marked difference in the composition of the repopulating human T cells in blood and spleen of mice treated with IL-2 or IL-10 alone compared to a combination of the two cytokines. Singly, both cytokines induced expansion of CD4+ as well as CD8+ T cells with a relatively diverse repertoire but in combination, they induced large oligoclonal expansions of CD4+ T cells with very limited Vβ repertoire and near complete absence of CD8+ T cells. Taken together, our results demonstrate for the first time that IL-10 directly contributes to GVHD pathogenesis. Further, when used in combination with IL-2, IL-10 has a major impact on the repertoire of the responding T cells.

Dysregulated cytokine production plays an important role in the clinical manifestation of acute graft-versus-host disease (aGVHD) following allogeneic bone marrow transplantation (BMT). The role of IL-10, anti-inflammatory cytokine in GVHD pathogenesis remains ambiguous. There are reports showing both beneficial and adverse effects of this cytokine in GVHD progression. Hence to determine the role of IL-10 in GVHD progression, we expressed hIL-10 in NOD-scid IL2rγcnull mice (NSG) by hydrodynamic gene delivery and evaluated its effect on xeogenic GVHD manifestations. We demonstrate that hIL-10 is capable of accelerating the hPBMC induced xeno-GVHD in NSG mice. The marked human T cell infiltration and tissue destruction observed in presence of IL-10 suggest that human IL-10 potently activates and expands pathogenic human T cells with the capacity to induce xeno GVHD. The significant reduction in the number of human cells at the initial phase and robust expansion at later stage suggests that those escapes IL-10 mediated suppression is responsible for the development of xeno-GVHD. Taken together, our results demonstrate for the first time that xenografted human T cells expand in response to IL-10 and mediate tissue damage in xeno GVHD mouse model. These finding provide caution against using rhIL-10 as an immunosuppressive cytokine after allogenic BMT.

Over expression cytokine 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 NOD/SCID/IL2Rγ-/- mice and siRNA treatment to inhibit HMGB1 release by human macrophages and dendritic cells dramatically reduces sepsis-induced mortality. Following CLP, as compared to 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 BLT mice) showed high serum levels of HMGB1 as well as multiple human, but not murine proinflammatory cytokines and uniformly succumbed, 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 AchR binding peptide (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 suppress a variety of human inflammatory diseases.

Regulatory T cells (TRegs) can suppress a wide range of immune cells, making them an ideal candidate for the treatment graft versus host disease (GVHD). The potential clinical translation of targeted therapy with in vitro expanded human nTRegs is being tested to prevent GVHD, which is a major cause for morbidity and mortality associated with hematopoietic stem cell transplantation. Their usefulness in therapy will depend on their capacity to survive, migrate and retain suppressive activity when introduced into a transplant recipient. The lack of a suitable animal model is a major impediment for investigating the behavior of adoptively transferred nTRegs in vivo. The aim of this study is to develop TRegs reconstituted humanized mice as a preclinical model for understanding their therapeutic usefulness in the transplantation setting. We show that injection of a plasmid encoding human IL-2 is necessary and sufficient for long term engraftment of in vitro expanded nTRegs in NOD-SCID IL2rγcnull mice. Furthermore, the reconstituted TRegs trafficked to different organs of the body and retained their characteristic suppressive function. Finally, in an IL-2 accelerated GVHD model, we show that these in vivo reconstituted TRegs are capable of preventing severe xenogenic response of human PBMCs. Thus, this novel hu-TReg mouse model offers a pre-clinical platform to study the in vivo function and stability of human nTRegs and their ability to modulate autoimmune diseases and GVHD.