Viral DNA was extracted from DF-1 cells mock infected or infected at 5 PFU/cell with MVA-WT, or MVA-LEO160-gp120. DNA-gp120/MVA-LEO160-gp120 induced an enhancement in the magnitude of gp120-specific CD4+ and CD8+ T-cell reactions, compared to DNA-gp120/MVA-B; with most of the reactions being mediated from the CD8+ T-cell compartment, having a T effector memory space phenotype. DNA-gp120/MVA-LEO160-gp120 also elicited a tendency to a higher magnitude of gp120-specific CD4+ T follicular helper cells, and moderate enhanced levels of antibodies against HIV-1 gp120. These findings revealed that this fresh optimized vaccinia disease promoter could be regarded as a promising strategy in HIV/AIDS vaccine design, confirming the importance Naproxen etemesil of early manifestation of heterologous antigen and its impact on the antigen-specific immunogenicity elicited by poxvirus-based vectors. = 5) received 100 g of DNA-gp120 (100 g of pCMV-gp120BX08) or 100 g of DNA-? (100 g of pCMV-?) in 50 L of PBS from the intramuscular (i.m.) route and 2 weeks later on received an intraperitoneal (i.p.) inoculation of 1 1 107 PFU of the related MVA disease (MVA-WT, MVA-B, or MVA-LEO160-gp120) in 200 L of PBS. Mice primed with sham DNA (DNA-?) and boosted with nonrecombinant MVA-WT were used like a control group. At 10 days after the last immunization, mice were sacrificed with carbon dioxide (CO2) and their spleens and blood samples were processed to measure the adaptive T cell and humoral immune responses to HIV-1 gp120, respectively, by using intracellular cytokine staining (ICS) assay or enzyme-linked immunosorbent assay (ELISA). Two impartial experiments were performed. 2.16. ICS Assay The magnitude, breadth, polyfunctionality, and phenotype of the HIV-1-specific T cell adaptive immune responses were analyzed by ICS as previously explained [34,37,38,39,43], with some modifications. After spleen processing, new 4 106 splenocytes (depleted of reddish blood cells) were seeded onto M96 plates and stimulated for 6 h in total RPMI 1640 medium supplemented with 10% FCS made up of 1 L/mL Golgiplug (BD Biosciences, Franklin Lakes, NJ, USA) to inhibit cytokine secretion; monensin Naproxen etemesil 1X (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA), anti-CD107aCFITC (BD Biosciences, Franklin Lakes, NJ, USA); and HIV-1 Env peptide pools (5 g/mL). Then, cells were washed, stained for the surface markers, fixed, permeabilized (Cytofix/Cytoperm kit; BD Biosciences, Franklin Lakes, NJ, USA), and stained intracellularly with the appropriate fluorochromes. Dead cells were excluded with the violet LIVE/DEAD stain kit (Invitrogen, Carlsbad, CA, USA). The fluorochrome-conjugated antibodies utilized for Naproxen etemesil functional analyses were CD3-phycoerythrin (PE)-CF594, CD4-allophycocyanin (APC)-Cy7, CD8-V500, IFN-CPE-Cy7, TNF-CPE, and IL-2CAPC. In addition, the antibodies utilized for phenotypic analyses were CD62L-Alexa 700 and CD127-peridinin chlorophyll protein (PerCP)-Cy5.5. All antibodies were from BD Biosciences, Franklin Lakes, NJ, USA. The magnitude of the HIV-1-specific T follicular helper (Tfh) Rabbit Polyclonal to SEC22B cell adaptive immune responses was analyzed by ICS as previously explained [44,45], with some modifications. After spleen processing, new, 4 106 splenocytes (depleted of reddish blood cells) were seeded onto M96 plates using RPMI-10% FCS and stimulated with 5 g/mL of Env peptide pools and 0.5 g/mL of Naproxen etemesil HIV-1 gp120 envelope protein from isolate BX08 (CNB) along with anti-CD154 Naproxen etemesil (CD40L)-PE antibody at 37 C. Two hours later, 1 L/mL protein transport inhibitor GolgiPlug (BFA, BD Biosciences, Franklin Lakes, NJ, USA), and monensin (1X; eBioscience, Thermo Fisher Scientific, Waltham, MA, USA), were added and cells were keep incubated for 4 additional hours at 37 C. Next, live cells were stained using fixable viability stain (FVS) 520 (BD Biosciences, Franklin Lakes, NJ, USA) for 20 min at 4 C. Then, after being washed twice with IB buffer (PBS 1X-FCS 2%-EDTA 2 mM), cells were stained for the surface markers using 50 L of the corresponding antibodies CD4-Alexa 700,.