Activating Natural Killer Cell Receptors, Selectins, and Inhibitory Siglecs Recognize Ebolavirus Glycoprotein

Expression of the extensively glycosylated Ebolavirus glycoprotein (EBOV-GP) induces physical alterations of surface molecules and plays a crucial role in viral pathogenicity. Here we investigate the interactions of EBOV-GP with host surface molecules using purified EBOV-GP, EBOV-GP-transfected cell lines, and EBOV-GP-pseudotyped lentiviral particles. Subsequently, we wanted to examine which receptors are involved in this recognition by binding studies to cells transfected with the EBOV-GP as well as to recombinant soluble EBOV-GP. As the viral components can also bind to inhibitory receptors of immune cells (e.g., Siglecs, TIM-1), they can even suppress the activity of immune effector cells. Our data show that natural killer (NK) cell receptors NKp44 and NKp46, selectins (CD62E/P/L), the host factors DC-SIGNR/DC-SIGN, and inhibitory Siglecs function as receptors for EBOV-GP. Our results show also moderate to strong avidity of homing receptors (P-, L-, and E-selectin) and DC-SIGNR/DC-SIGN to purified EBOV-GP, to cells transfected with EBOV-GP, as well as to the envelope of a pseudotyped lentiviral vector carrying the EBOV-GP. The concomitant activation and inhibition of the immune system exemplifies the evolutionary antagonism between the immune system and pathogens. Altogether these interactions with activating and inhibitory receptors result in a reduced NK cell-mediated lysis of EBOV-GP-expressing cells. Modulation of these interactions may provide new strategies for treating infections caused by this virus.publishedVersio

Prophylactic DNA vaccine targeting Foxp3 + regulatory T cells depletes myeloid-derived suppressor cells and improves anti-melanoma immune responses in a murine model

Regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) are the two important and interactive immunosuppressive components of the tumor microenvironment that hamper anti-tumor immune responses. Therefore, targeting these two populations together might be beneficial for overcoming immune suppression in the tumor microenvironment. We have recently shown that prophylactic Foxp3 DNA/recombinant protein vaccine (Foxp3 vaccine) promotes immunity against Treg in tumor-free conditions. In the present study, we investigated the immune modulatory effects of a prophylactic regimen of the redesigned Foxp3 vaccine in the B16F10 melanoma model. Our results indicate that Foxp3 vaccination continuously reduces Treg population in both the tumor site and the spleen. Surprisingly, Treg reduction was associated with a significant decrease in the frequency of MDSC, both in the spleen and in the tumor environment. Furthermore, Foxp3 vaccination resulted in a significant reduction of arginase-1(Arg-1)-induced nitric oxide synthase (iNOS), reactive oxygen species (ROS) and suppressed MDSC activity. Moreover, this concurrent depletion restored production of inflammatory cytokine IFN-γ and enhanced tumor-specific CTL response, which subsequently resulted in the reduction of tumor growth and the improved survival rate of vaccinated mice. In conclusion, our results revealed that Foxp3 vaccine promotes an immune response against tumor by targeting both Treg and MDSC, which could be exploited as a potential immunotherapy approach.. © 2017, Springer-Verlag GmbH Germany, part of Springer Nature

Prophylactic DNA vaccine targeting Foxp3+regulatory T cells depletes myeloid-derived suppressor cells and improves anti-melanoma immune responses in a murine model

Abstract Regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) are the two important and interactive immunosuppressive components of the tumor microenvironment that hamper anti-tumor immune responses. Therefore, targeting these two populations together might be beneficial for overcoming immune suppression in the tumor microenvironment. We have recently shown that prophylactic Foxp3 DNA/recombinant protein vaccine (Foxp3 vaccine) promotes immunity against Treg in tumor-free conditions. In the present study, we investigated the immune modulatory effects of a prophylactic regimen of the redesigned Foxp3 vaccine in the B16F10 melanoma model. Our results indicate that Foxp3 vaccination continuously reduces Treg population in both the tumor site and the spleen. Surprisingly, Treg reduction was associated with a significant decrease in the frequency of MDSC, both in the spleen and in the tumor environment. Furthermore, Foxp3 vaccination resulted in a significant reduction of arginase-1(Arg-1)-induced nitric oxide synthase (iNOS), reactive oxygen species (ROS) and suppressed MDSC activity. Moreover, this concurrent depletion restored production of inflammatory cytokine IFN-γ and enhanced tumor-specific CTL response, which subsequently resulted in the reduction of tumor growth and the improved survival rate of vaccinated mice. In conclusion, our results revealed that Foxp3 vaccine promotes an immune response against tumor by targeting both Treg and MDSC, which could be exploited as a potential immunotherapy approach. Keywords Regulatory T cells Myeloid-derived suppressor cells Foxp3 Melanom

Cloning, expression and purification of Ag85A-ESAT6 antigens of Mycobacterium tuberculosis as a fusion protein

Aims and objectives: Secretory antigens of Ag85 complex and 6-kDa Early Secreted Antigenic Target (ESAT-6) play a role in cell wall synthesis and virulence of Mycobacterium tuberculosis (MTB), respectively. They could induce a potent immune response and showed protective effects in some studies in animal models. So, they are considered as potential candidate antigens in new vaccination strategies against MTB. The aim of this study includes cloning, expression and purification of recombinant Ag85A-ESAT6 as a fusion protein for further studies of vaccine designation. Materials and Methods: A synthetic DNA fragment composed of coding sequences of Ag85A and ESAT6 was cloned into the BamHI and EcoRI restriction site of pET28a plasmid. Then the recombinant plasmid was transformed to Escherichia coli BL21 (DE3) and expressed in optimum condition. The expressed protein was analyzed and confirmed by SDS–PAGE and western blotting using anti-His tag and monoclonal anti-ESAT6 antibodies. Protein purification was performed by Ni–NTA spin column under denaturing conditions. Results: The results of SDS–PAGE and western blotting showed that Ag85A-ESAT6 recombinant fusion protein was successfully cloned and expressed with a His tag in its N-terminal. Considering the expression of this protein as an inclusion body, its purification was done by denaturing protocol. Conclusion: The Ag85A-ESAT6 fusion protein expressed in this study can be combined with a suitable adjuvant to induce the cellular and humoral immune responses, and its protective effects against MTB could be evaluated in subsequent studies

High avidity anti-integrase antibodies discriminate recent and non-recent HIV infection: Implications for HIV incidence assay

Estimation of HIV incidence provides real-time information of HIV transmission trends for decision makers. Anti-integrase antibodies are the last ones produced during seroconversion and presence of high-avidity anti-integrase antibodies indicates the chronicity of HIV infection. This study aimed to evaluate the performance of these antibodies in discriminating of recent from non-recent HIV infection. For this purpose, different ELISA formats were developed to detect high-avidity anti-integrase antibodies in a commercially available performance panel, and the best assay was selected for further evaluation. The false recent rate of the selected assay was evaluated in a panel of Iranian patients and compared to two commercial assays, BED-EIA and LAg-Avidity. While the false recent rate of the developed assay was 3.8, it was 14.1 and 1.3 for BED-EIA and LAg-Avidity, respectively. To our knowledge, this is the first report to study the performance of high-avidity anti-integrase antibodies for classification of HIV infection. The preliminary results showed that the specificity of the newly developed assay is markedly higher than BED-EIA and is comparable with LAg-Avidity. The promising results point to the potential use of anti-integrase antibodies as a biomarker in HIV incidence laboratory tests or algorithms. The developed assay needs further evaluation in future. © 201

Development of an integrase-based ELISA for specific diagnosis of individuals infected with HIV

Currently, enzyme immunoassays (EIAs) are the most common immunological diagnostic methods that are used as the screening tool in HIV detection. Among all three major genes of HIV, the products of gag and env are usually used in EIAs (ELISAs and rapid tests). Hence, the presence of cross reacting antibodies against these antigens leads to the appearance of repetitive false positive results in screening tests. Re-testing the primary reactive samples with EIAs using other HIV antigens can considerably reduce the rate of false positive results. The products of pol gene may act as an appropriate candidate in this context. Integrase is a conserved and immunogenic product of HIV, encoded by the pol gene. The aim of this research was to determine the sensitivity and specificity of an ELISA detecting integrase antibodies. Recombinant integrase was produced in Escherichia coli to develop the integrase-based ELISA. Assay performance was evaluated by HIV positive and negative sera and an HIV panel of BBI (PRB-601). The sensitivity and specificity of assay was determined as 96.7 95% confidence interval: 91.3-98.9% and 100% 95% CI: 96.1-100%, respectively. High specificity of this assay may suggest its possible use in the detection of HIV. © 2015 Elsevier B.V

High avidity anti-integrase antibodies discriminate recent and non-recent HIV infection: Implications for HIV incidence assay

Estimation of HIV incidence provides real-time information of HIV transmission trends for decision makers. Anti-integrase antibodies are the last ones produced during seroconversion and presence of high-avidity anti-integrase antibodies indicates the chronicity of HIV infection. This study aimed to evaluate the performance of these antibodies in discriminating of recent from non-recent HIV infection. For this purpose, different ELISA formats were developed to detect high-avidity anti-integrase antibodies in a commercially available performance panel, and the best assay was selected for further evaluation. The false recent rate of the selected assay was evaluated in a panel of Iranian patients and compared to two commercial assays, BED-EIA and LAg-Avidity. While the false recent rate of the developed assay was 3.8, it was 14.1 and 1.3 for BED-EIA and LAg-Avidity, respectively. To our knowledge, this is the first report to study the performance of high-avidity anti-integrase antibodies for classification of HIV infection. The preliminary results showed that the specificity of the newly developed assay is markedly higher than BED-EIA and is comparable with LAg-Avidity. The promising results point to the potential use of anti-integrase antibodies as a biomarker in HIV incidence laboratory tests or algorithms. The developed assay needs further evaluation in future. © 201

Design and constructing pBGGT Plasmid: a carrier plasmid for Betathalassaemia gene targeting

Background: Most of molecular biology studies depend on making gene constructs. Although commercial plasmids are widely used for this purpose, sometimes due to the nature of the restriction sites or need of multiple subcloning, usual restriction sites available in original multiple cloning sites (MCS) of the plasmids could not be easily used, if not impossible at all. Here, we report an easy and fast method to construct suitable plasmid with a new MCS for constructing a 16kb gene targeting plasmid.Methods: Firstly, the suitable MCS was designed by studying the sequence of desired gene fragments in Gene runner software. Two partial complementary 74 base long oligonucleotides were designed and constructed to make this MCS. The original pUC19 MCS was replaced with the new one by enzymatic digestion of the plasmid and removal of the MCS, followed by adding the two complementary oligonucleotides and ligating the construct and transforming it into Ecoli TOP10 F'. The new plasmid was then purified and sequenced by M13 forward and reverse primers.Findings: Synthesis of two 74 base polynuclotides was successful, and these polynucleotides formed a double stranded fragment which was successfully inserted between HindIII-EcoRI sites of pUC19. Analysis of intermediate step results showed successful progress of cloning reaction. Final analysis of the plasmid by restriction analysis and sequencing the MCS confirmed authenticity of the new plasmid.Conclusions: The method described here is a fast and easy way to make suitable plasmid out of commercially available plasmids. This process can considerably decrease the time and cost of plasmid construction. Availability of suitable restriction sites in proper order makes it possible to directionally clone the desired gene fragments which is more efficient and excludes screening steps for the right direction of the fragments. The plasmid reported herein is specifically tailored to insert different fragments of a beta-globin gene targeting construct