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Background:Using molecular adjuvants offers an attractive strategy to augment DNA vaccine-mediated immune responses. Several studies have revealed that an efficient HCV vaccine model should be able to induce both humoral and cell mediated immune responses targeting the conserved regions of the virus to circumvent the immune escape mutants. The beta chemokine Macrophage Inflammatory Protein 3-beta (MIP-3beta) is a key modulator of dendritic cells (DCs) and T-cells interaction, functions during immune response induction and is secreted specifically by cells in the lymphoid tissues.Objectives:In the present study, we questioned whether co-administration of MIP-3beta gene could enhance the immune responses to HCV core in DNA vaccination.Materials and Methods:Expression and biological activity of MIP-3beta expressing plasmid were evaluated by ELISA and transwell migration assays, respectively. HCV core DNA vaccine ± plasmid expressing MIP-3beta were electroporated subcutaneously to the front foot pads of BALB/c mice on days 0 and 14, and HCV core protein booster was applied to all core-DNA-vaccine received mice on the day 28. Both cell mediated immunity (proliferation, IFN-γ and IL-4 cytokine release, IFN-γ ELISpot and cytotoxic Granzyme B release assays) and humoral immune responses (total IgG and IgG2a/IgG1 subtyping) were evaluated ten days after final immunization.Results:Mice covaccinated with MIP-3beta elicited an enhanced Th1 biased systemic immune response as evidenced by higher IFN-γ/IL-4 and anti-core IgG2a/IgG1 ratio, lymphoproliferation, strong cytolytic GrzB release and enhanced population of IFN-γ producing immunocytes. Likewise, the humoral immune response assumed as the total anti-core IgG level was augmented by MIP-3beta co-delivery.Conclusions:These results exhibited the immuno potentiator effects of MIP-3beta plasmid when coadministrated with the HCV core DNA vaccine. Complimentary studies integrating MIP-3beta as a genetic adjuvant in HCV-core-DNA vaccination models are warranted.

Summary. -Despite the success of highly active antiretroviral therapy, AIDS still remains as one of the most important world health problems. Toxicity of current available drugs and inevitable emergence of multi-drug resistant strains makes things worse. In the present study a series of novel Biginelli-type pyrimidine compounds were evaluated as potential anti-human immunodefi ciency virus (HIV)-1 agents using green fl uorescence protein (GFP) reporter single round HIV-1 infection assay. Th e rate of infected cells was monitored by fl owcytometry. Th e eff ect of compounds on the cellular proliferation was considered as the cyotoxicity. Th e anti-HIV-1 active compounds were selected for HIV-1 replication and syncytium formation assays. Th e antiretroviral activity of compounds was measured against luciferase reporter A murine leukemia virus (AMLV) virions as the retrovirus control. Compounds 2, 5, 6, 8, 11, 12, 13, 17, 18, 20, and 21 were the most potent against HIV-1. Compound 8 had the 50% inhibitory concentration (IC 50 ) of 100 nmol/l for inhibiting HIV-1 replication and 50% cytotoxic concentration (CC 50 ) was up to 100 μmol/l (therapeutic index (TI) >1000). Results show that the active compounds were able to inhibit the retrovirus control as well. Analysis of structure of the studied compounds proved relationships with their anti-HIV-1 eff ects. Some of the studied compounds seem to be promising anti-HIV-1 drug candidates. Structural manipulation based on the well-defi ned structure-activity relationships might propose some new leads for anti-HIV-1 drug discovery programs.Keywords: antivirals; HIV-1; Biginelli-type pyrimidines * Co-corresponding authors. E-mail: fassihi@pharm.mui.ac.ir, mrasadeghi@pasteur.ac.ir; phone: +98-311-7922562, +98-21 66468765.Abbreviations: AMLV = A murine leukemia virus; DHPMs = 3,4-dihydropyrimidine-2(1H)-ones; ENV(s) = envelope glycoprotein(s); GFP = green fl uorescence protein; HAART = highly active antiretroviral therapy; HIV = human immunodefi ciency virus; IN = integrase; RT = reverse transcriptase; RTU = reverse transcriptase unit; SCR = single-cycle replicable; IC 50 = 50% inhibitory concentration; CC 50 = 50% cytotoxic concentration; SI = selectivity index (CC 25 / IC 25 ); TI = therapeutic index (CC 50 /IC 50 ); VSVG = vesicular stomatitis virus-glycoprotein

Plasmid DNA vaccination is a promising vaccine platform for prevention and treatment of infectious disease. Enhancement of the DNA vaccine potency by co-inoculation of immunoadjuvant has been shown to be an effective strategy. Modulation of dendritic cells and T-cells locomotion and trafficking to prime an immune response is mediated by distinct chemokines. The recent study was designed to elucidate the adjuvant activity of plasmid expressing CC-chemokine ligand 20 (pCCL20) in co-inoculation with hepatitis C virus (HCV) core DNA vaccine immunization. pCCL20 was constructed and evaluated for its functional expression. Sub-cutaneous inoculation of pCCL20 with HCV core DNA vaccine was performed via electroporation in BALB/c mice on day 0 and 14 and a HCV core protein booster was applied on day 28. On week after final immunization, both humoral and cell-mediated immune responses were assessed by indirect ELISA for core specific antibodies, lymphocyte proliferation, cytokine ELISA/ELISpot and cytotoxic Grenzyme B (GrzB) release assays. Mice were co-immunized with pCCL20 developed higher levels of core specific IFN-γ/IL-4 ratio and IL-2 release, IFN-γ producing cells, lymphocyte proliferation and cytotoxic Grenzyme B release in both draining lymph nodes and spleen cells of immunized mice. The core-specific serum total IgG and IgG2a/IgG1 ratio were significantly higher when the pCCL20 was co-inoculated. These results suggest the potential of CCL20 chemokine as vaccine adjuvant to enhance Th1 mediated cellular and humoral immune responses in HCV core DNA immunization.

Cytokines are mediators for polarization of immune response in vaccines. Studies show that co-immunization of DNA vaccines with granulocyte-macrophage colony-stimulating factor (GM-CSF) can increase immune responses. Here, experimental mice were immunized with HIV-1 DNA vaccine with GM-CSF and boosted with recombinant vaccine. Lymphocyte proliferation with Brdu and CTL activity, IL-4, IFN-γ, IL-17 cytokines, total antibody, and IgG1 and IgG2a isotypes were assessed with ELISA. Results show that GM-CSF as adjuvant in DNA immunization significantly increased lymphocyte proliferation and IFN-γ cytokines, but CTL response was tiny increased. Also GM-CSF as adjuvant decreased IL-4 cytokine vs mere vaccine group. IL-17 in the group that immunized with mixture of DNA vaccine/GM-CSF was significantly increased vs DNA vaccine group. Result of total antibody shows that GM-CSF increased antibody response in which both IgG1 and IgG2a increased. Overall, results confirmed the beneficial effect of GM-CSF as adjuvant to increase vaccine immunogenicity. The hallmark result of this study was to increase IL-17 cytokine with DNA vaccine/GM-CSF immunized group. This study for the first time provides the evidence of the potency of GM-CSF in the induction of IL-17 in response to a vaccine, which is important for control of infection such as HIV-1.

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