Granulocyte-macrophage colony-stimulating factor, a potent adjuvant for polarization to Th-17 pattern: an experience on HIV-1 vaccine model.

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-1tat/pol/gag/env 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.

Modulation of hepatitis C virus core DNA vaccine immune responses by co-immunization with CC-chemokine ligand 20 (CCL20) gene as immunoadjuvant.

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.

Enhancement of Immune Responses by Co-delivery of CCL19/MIP-3beta Chemokine Plasmid With HCV Core DNA/Protein Immunization.

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.

Evaluation of optimal concentration and exposure duration of valproic acid alone or in combination with ViraDuctin to augment adenovirus transduction in human adipose stem cells.

BACKGROUND AIMS: Recombinant adenoviruses have tremendous potential in both gene therapy research and therapeutic applications. Mesenchymal stromal cells have a set of several properties that make them ideally suited for both regenerative medicine and gene and drug delivery. A limitation of adenoviral-mediated gene transfer is indeed the poor transduction rate of cells with low or no levels of the specific adenoviral cell surface receptor coxsackie virus and adenovirus receptor (CAR), such as human mesenchymal stromal cells. In the present work, we tried to increase the adenovirus transduction level and mediated gene delivery of human adipose stem cells with the use of valproic acid (VPA) and determined the proper concentration and duration of treatment alone or in combination with ViraDuctin adenovirus transduction reagent. METHODS: Green fluorescent protein-expressing recombinant adenovirus was propagated. The effects of various doses and exposure periods of VPA on CAR expression in human adipose stem cells were speculated by quantitative real-time polymerase chain reaction and adenoviral transduction rate by flow cytometry in different doses and time intervals of VPA and in combination with ViraDuctin transduction reagent. RESULTS: CAR messenger RNA upregulation through VPA was observed in human adipose stem cells; it was a dependent factor of dose and exposure time. Consequently, adenoviral transduction level of human adipose stem cells treated with VPA was increased, and co-administration of VPA and ViraDuctin further enhanced the transduction rate. CONCLUSIONS: These results confirm that addition of VPA to hASCs alone or in combination with ViraDuctin has enhancing effects on adenoviral transduction rate, which can be auspicious in adenoviral-mediated gene therapy.

ELISPOT analysis of a new CTL based DNA vaccine for HIV-1 using GM-CSF in DNA prime/peptide boost strategy: GM-CSF induced long-lived memory responses.

Genetic adjuvants have potential role in improvement of immune responses against DNA vaccines. GM-CSF as a genetic adjuvant can recruit and augment dendritic cell numbers in the site of immune responses and thereby induce cellular and humoral immune responses. Here we show that co-immunization of a DNA vaccine from HIV-1P24-Nef with GM-CSF in DNA priming and peptide boost strategy increases the immunogenicity of our candidate vaccine. Analysis of immune response shows that co-immunization with GM-CSF boosts cellular immune responses through increasing proliferation activity and CTL function. Results of cytokine profile studies show that both IL-4 and IFN-γ levels were augmented. Also, co-immunization with GM-CSF resulted in a higher level of total IgG, comprising approximately equal levels of both specific IgG1 and IgG2a subtypes. Monitoring of cellular and humoral immune responses for 20 weeks after final immunization revealed the aptitude of GM-CSF for inducing long-lived humoral and cell mediated immune responses. Overall, our results suggest that GM-CSF is able to induce long term memory for the HIV-1 P24-Nef vaccine candidate while the exact mechanisms involved remained to be clarified.

Effect of immunological adjuvants: GM-CSF (granulocyte-monocyte colony stimulating factor) and IL-23 (interleukin-23) on immune responses generated against hepatitis C virus core DNA vaccine.

The use of cytokines as adjuvants has been shown to be a promising approach for enhancing DNA vaccine induced-immune responses. In this report, we investigate the administration of cytokines to modulate both humoral and cell-mediated immune responses elicited by an HCV-core plasmid DNA vaccine in Balb/c mice. Our studies indicate that the HCV-core DNA vaccine has been able to induce both antibody and cellular immunity in a DNA prime-protein boost regimen. GM-CSF (granulocyte-monocyte colony stimulating factor) which is considered to be a cytokine displaying both Th1 and Th2 characteristics, and plays an important role in augmenting antibody and cell-mediated immunity was also administered. The induction of cellular immunity was not as striking as humoral immunity in this case. To obtain a stronger cellular response, IL-23, a Th1 cytokine belonging to the IL-12 family, was also included in the regimen. Spleen cell proliferation, IFN-gamma production from spleen cells and specific serum IgG2a, all demonstrate the enhancement of cell-mediated immunity without any observable suppressive effect on antibody and humoral immune responses. We also examined the timing of plasmid IL-23 administration on the phenotype of the resultant T cell responses in a 3 day interval, before and after plasmid GM-CSF administration. The results did not indicate any change in the Th1/Th2 balance as compared with simultaneous IL-23 administration.