Increased Plasma Levels of Soluble CD27 among HIV/HCV Co-infected and HIV/HCV/GBV-C Triply Infected Subjects

CD27 is a biomarker associated with both T-cells and B-cells activation .Plasma soluble CD27 (sCD27) was identified as  a marker of disease outcome in Human Immunodeficiency Virus (HIV) infection .Testing of plasma sCD27 represents a good tool to monitor the change of immune activation during HIV infection.We sought to analyses role of Hepatitis C Virus (HCV) and also GB Virus type C (GBV-C) co-infections on HIV-related immune activation, through measuring sCD27 plasma levels.Blood samples from a total of 86 patients with HIV infection were taken. Plasmas were analyzed for HCV using serologic test and GBV-C by reverse transcriptase polymerase chain reaction (RT-PCR). CD4+ and CD8+T-cell counts were evaluated by CD3/CD4+ and CD3/CD8+ double staining of whole blood followed by flow cytometric analysis .Then  Cross-sectional comparison of sCD27 plasma levels was carried out among patients : HIV (n=20), HIV/ GBV-C (n=14), HIV/ (HCV) (n=26) and HIV/HCV/GBV-C (n=26).Plasma level of sCD27 was higher in HIV/HCV/GBV-C patients as compared to HIV mono-infected patients (p= 0.006) and based on results there was significant differences in the plasma levels of sCD27 between HIV-infected individuals with and without HCV coinfection (P=0.017) and also correlation between sCD27 and percent of CD4+T-cells was in highest level among HIV/HCV co-infected patients group [r= -0.59 (p=0.001)]. High levels of sCD27 among HIV/HCV patients argues in favor of sCD27 plasma level determination for monitoring of clinical features among HIV/HCV coinfected patients

Expression of Leishmania major LmSTI1 in Yeast Pichia Pastoris

Background: Leishmania major LmSTI1 is a conserved protein among different species of leishmania, and expressed in both amastigote and promastigote forms of L. major life cycle. It has previously been expressed in bacterial systems.Materials and Methods: To express LmSTI1 in the methylotrophic yeast         Pichia pastoris (P. pastoris), the shuttle vector pPICZA containing gene lmsti1 was constructed under the control of the AOX1 promoter. The recombinant vector was electro-transformed into P. pastoris, and induced by 0.5% methanol in the buffered medium. The expression of the LmSTI1 protein was visualized in the total soluble protein of P. pastoris by 12% SDS-PAGE, and further confirmed by Western blotting with L.major-infected mouse sera and HRP-conjugated goat anti-mouse IgG as the first and secondary antibodies, respectively.Results: The expression level was 0.2% of total soluble proteins.Conclusion: It might be possible to use this formulation as a whole yeast candidate vaccine against cutaneous leishmanization

The Impact of CRISPR-Cas System on Antiviral Therapy

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein nuclease (Cas) is identified as an adaptive immune system in archaea and bacteria. Type II of this system, CRISPR-Cas9, is the most versatile form that has enabled facile and efficient targeted genome editing. Viral infections have serious impacts on global health and conventional antiviral therapies have not yielded a successful solution hitherto. The CRISPR-Cas9 system represents a promising tool for eliminating viral infections. In this review, we highlight 1) the recent progress of CRISPR-Cas technology in decoding and diagnosis of viral outbreaks, 2) its applications to eliminate viral infections in both pre-integration and provirus stages, and 3) various delivery systems that are employed to introduce the platform into target cells

MAIT cells launch a rapid, robust and distinct hyperinflammatory response to bacterial superantigens and quickly acquire an anergic phenotype that impedes their cognate antimicrobial function: Defining a novel mechanism of superantigen-induced immunopathology and immunosuppression

Superantigens (SAgs) are potent exotoxins secreted by Staphylococcus aureus and Streptococcus pyogenes. They target a large fraction of T cell pools to set in motion a "cytokine storm" with severe and sometimes life-threatening consequences typically encountered in toxic shock syndrome (TSS). Given the rapidity with which TSS develops, designing timely and truly targeted therapies for this syndrome requires identification of key mediators of the cytokine storm's initial wave. Equally important, early host responses to SAgs can be accompanied or followed by a state of immunosuppression, which in turn jeopardizes the host's ability to combat and clear infections. Unlike in mouse models, the mechanisms underlying SAg-associated immunosuppression in humans are ill-defined. In this work, we have identified a population of innate-like T cells, called mucosa-associated invariant T (MAIT) cells, as the most powerful source of pro-inflammatory cytokines after exposure to SAgs. We have utilized primary human peripheral blood and hepatic mononuclear cells, mouse MAIT hybridoma lines, HLA-DR4-transgenic mice, MAIThighHLA-DR4+ bone marrow chimeras, and humanized NOD-scid IL-2Rγnull mice to demonstrate for the first time that: i) mouse and human MAIT cells are hyperresponsive to SAgs, typified by staphylococcal enterotoxin B (SEB); ii) the human MAIT cell response to SEB is rapid and far greater in magnitude than that launched by unfractionated conventional T, invariant natural killer T (iNKT) or γδ T cells, and is characterized by production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2, but not IL-17A; iii) high-affinity MHC class II interaction with SAgs, but not MHC-related protein 1 (MR1) participation, is required for MAIT cell activation; iv) MAIT cell responses to SEB can occur in a T cell receptor (TCR) Vβ-specific manner but are largely contributed by IL-12 and IL-18; v) as MAIT cells are primed by SAgs, they also begin to develop a molecular signature consistent with exhaustion and failure to participate in antimicrobial defense. Accordingly, they upregulate lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin-3 (TIM-3), and/or programmed cell death-1 (PD-1), and acquire an anergic phenotype that interferes with their cognate function against Klebsiella pneumoniae and Escherichia coli; vi) MAIT cell hyperactivation and anergy co-utilize a signaling pathway that is governed by p38 and MEK1/2. Collectively, our findings demonstrate a pathogenic, rather than protective, role for MAIT cells during infection. Furthermore, we propose a novel mechanism of SAg-associated immunosuppression in humans. MAIT cells may therefore provide an attractive therapeutic target for the management of both early and late phases of severe SAg-mediated illnesses

Prevalence of HIV?1 pre?treatment drug resistance in a southernprovince of Iran, 2016?2017

Abstract HIV-1 transmitted drug resistance (TDR)occurs when primary viruses bear drug resistance mutations(DRMs). TDR causes first-line antiretroviral (ARV)therapy (ART) failure and is becoming more pronounceddue to the widespread use of ART. With the absence ofroutine individual-level drug resistance testing, the WorldHealth Organization (WHO) recommends the tracking ofTDR mutations and optimizing the first-line ART followingpre-treatment drug resistance (PDR) surveys. Here, wereport the PDR frequency for the first time in Hormozgan,a southern province of Iran. In this study, 41 blood samplesfrom HIV-1-positive ART-candidate volunteers werecollected across the province between April 2016 andMarch 2017. Phylogenetic analysis of the sequenced protease(PR) and reverse transcriptase (RT) regions showedthat 39 out of 41 samples (95%) were CRF35_AD and thetwo remaining cases were subtype B (2.5%) and C (2.5%).D67G (2.4%), a mutation that reduces susceptibility to nucleoside reverse transcriptase inhibitors (NRTIs) was theonly detectable TDR mutation in this population. Two otherDRMs, including E138A (9.7%) and V179T (4.9%), whichconfer resistance to non-nucleoside reverse transcriptaseinhibitors (NNRTIs), were also identified. Although nomajor protease inhibitor (PI) resistance mutations weredetected, the minor mutations L10F and L33F (2.5% each)as well as several highly frequent polymorphic mutationswere identified. Our results show a PDR frequency of 17%in infected individuals from Hormozgan, classified furtheras 2.4% NRTIs and 14.6% NNRTIs. These results suggestthat first-line ART should be practiced carefully in Hormozganprovince, and alternative regimens may becomenecessary for all starters

Designing, Constructing and Immunogenic Evaluation of Polytope DNA Constructs by the Application of Hepatitis C Virus Immunodominant Epitopes in BALB/c Mouse

Objective: Polytope DNA vaccines, capable of focusing the cytotoxic T lymphocyte (CTL)response on critical epitopes, represent a promising approach in HCV immunotherapy. Nevertheless,due to controversial rules governing epitope processing and the low level expression/immunogenicity of recombinant polytope peptides, designing and primary expression/immunogenicity analysis of these vaccine types should be the first consideration prior tocostly transgenic animal studies.Materials and Methods: Four HLA-A2 and H-2d restricted CTL epitopes were selected anddesigned in three appropriate sequential tandems based on epitope and proteasomal cleavagepredictions. The related nucleotide sequences were synthesized using SOEing PCRmethod and cloned into a pcDNA3.1 vector, either alone or fused to the small hepatitis B surfaceantigen (HBsAg-S) gene. Following the preparation of polyclonal anti-sera, expression/secretion of polytopes was evaluated in Cos-7 cells by using immunofluorescence, Westernblot,dot blot, ELISA and RT-PCR techniques. The immunogenicity of the plasmids was alsoassessed through the delayed-type hypersensitivity (DTH) assay in BALB/c mice.Results: Due to in silico designs and optimizations, the polytope products of constructedplasmids were efficiently detected in vitro through common techniques and HBsAg-S-basedparticles were shown to be secreted into the culture media (up to 30%). Moreover, all plasmidswere able to efficiently induce a positive DTH response while HBsAg-S fusion constructsindicated a significant immunopotential effect towards the incorporated mouse epitopes.Conclusion: Designed polytope constructs of this study are efficiently expressed and processed.They have the required initial potency for further immunogenicity analysis in transgenicmice

Toward the Development of a Single-Round Infection Assay Based on EGFP Reporting for Anti-HIV-1 Drug Discovery

Background: The rapid increase of HIV-1 strains resistant to current antiretroviral drugs is a challenge for successful AIDS therapy. This necessitates the development of novel drugs, and to this end, availability of screening systems for in vitro drug discovery is a priority. Herein, we report the modification of a previously developed system for increased sensitivity, ease of use, and cost-efficiency, based on the application of the EGFP marker. Methods: A PCR-amplified gfp gene (gfp) was cloned into pmzNL4-3, the plasmid already designed to produce single-cycle replicable virions, in frame with the reverse-transcriptase gene to construct the pmzNL4-3/GFP plasmid. GFP-mzNL4-3 pseudo-typed virions, as the first progeny viruses, were recovered from the culture supernatant of HEK293T cells co-transfected with pmzNL4-3/GFP and the helper plasmids pSPAX2 and pMD2G, which respectively encode HIV-1 Gag-Pol and vesicular stomatitis virus glycoprotein. Single-cycle replication and virion production were assessed by syncytia formation, p24 antigen assays, and electron and fluorescence microscopy. Results: The incorporation of EGFP into the viral particles allowed their quantification by fluorometry, flow-cytometry, and fluorescence microscopy; however, this modification did not affect the single-round infectivity or production rate of the GFP fluorescence-emitting virions. Conclusions: Our results certify the development of a rapid, inexpensive, and safe GFP-reporting single-cycle replicable system for anti-HIV drug discovery. Further experiments are needed to measure the validity and robustness of the assay

Evaluation of cellular responses for a chimeric HBsAg-HCV core DNA vaccine in BALB/c mice

Background: Fusion of Hepatitis B virus surface antigen (HBsAg) to a DNA construct might be considered as a strategy to enhance cellular and cytotoxic T-lymphocytes (CTL) responses of a Hepatitis C Virus core protein (HCVcp)-based DNA vaccine comparable to that of adjuvanted protein (subunit) immunization. Materials and Methods: pCHCORE vector harboring coding sequence of HBsAg and HCVcp (amino acids 2-120) in tandem within the pCDNA3.1 backbone was constructed. The corresponding recombinant HCVcp was also expressed and purified in Escherichia coli. Mice were immunized either by adjuvanted HCVcp (pluronic acid + protein) or by pCHCORE vector primed/protein boosted immunization regimen. The cellular immune responses (proliferation, In vivo CTL assay and IFN-g/IL-4 ELISpot) against a strong and dominant H2-d restricted, CD8 + -epitopic peptide (C39) (core 39-48; RRGPRLGVRA) of HCVcp were compared in immunized animals. Result: Proper expression of the fused protein by pCHCORE in transiently transfected HEK 293T cells and in the expected size (around 50 kDa) was confirmed by western blotting. The immunization results indicated that the pCHCORE shifted the immune responses pathway to Th1 by enhancing the IFN-g cytokine level much higher than protein immunization while the proliferative and CTL responses were comparable (or slightly in favor of DNA immunization). Conclusion: Fusion of HBsAg to HCVcp in the context of a DNA vaccine modality could augment Th1-oriented cellular and CTL responses toward a protective epitope, comparable to that of HCVcp (subunit HCV vaccine) immunization