m.) at the gastrocnemius muscle at a dose of 109 viral particles (vp) in a total volume of 50 μl (i.e., 25 μl in each leg). Boosting immunizations were given 4-week post-priming in the same procedure as above in all cases. The BCG-CS and Ad35-CS constructs, expressing CSp, have been described previously [6] and [18]. The immunization design and the dosage of the different vaccines
are summarized in Table 1. Specific responses to P. falciparum CSp were measured by stimulating splenocytes and LLPCs with peptides deduced from the CSp antigen; Selleck VE 821 namely, the C-terminal (C-CSp, PfCS282-383), N-terminal (N-CSp, PfCS22-110) and immunodominant CD8+ T cell epitope (IDE-CSp, PfCS-NYDNAGTNL). The synthesis and immunological characterizations of those peptides have been reported in details elsewhere [19] and [20]. The rCSp was provided by Crucell (Leiden, The Netherlands) and has been described elsewhere [12]. Spleen-cell suspensions were prepared by teasing the organ with sterile forceps followed by passing through 27G needles several times, and then centrifugation. Bone marrow (BM) cells were collected from the BM of femurs and tibias by flushing them with RPMI. Red
blood cells (RBC) were removed by resuspending cells in ACK RBC-lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA in dH2O and adjusted pH to 7.2–7.4 http://www.selleckchem.com/products/gw3965.html with 1 M HCl; all compounds were purchased from Sigma–Aldrich, Steinheim, Germany) for 5 min before adding excess of RPMI. Splenocytes and LLPCs found were purified by centrifugation and resuspended in complete RPMI (RPMI 1640, 10% FCS,
100 IU/ml penicillin, 100 mg/ml streptomycin, 4 mM l-glutamine). CS-specific antibody responses were assessed by ELISA. Ninety-six-well microtiter plates (Costar 96-well HB half Area plate, Corning Inc, NY) were coated overnight with 2 μg/ml CSp in 0.05 M carbonate buffer (pH 9.6) at room temperature. Plates were washed three times with PBS/0.05% Tween 20 and a 1:400-dilution of individual serum samples were added to corresponding wells and a serial dilution of 2-fold with PBS/0.05% Tween 20. Plates were incubated for 2 h at room temperature and were washed three times and incubated with alkaline phosphatase-labeled anti-mouse IgG (Southern Biotech, Birmingham, AL, USA). For detection of IgG subclasses, samples were incubated with alkaline phosphatase-labeled anti-mouse IgG1 or IgG2a antibodies (Southern Biotech, Birmingham, AL). The enzyme/substrate reaction was developed using p-nitrophenyl phosphate (Sigma–Aldrich, Steinheim, Germany). Optical density was measured at 405 nm by using a V max ELISA reader (Molecular Devices Instruments). CSp-specific cellular immune responses in vaccinated mice were measured using an IFN-γ ELISPOT assay. The splenocytes from each group of mice were stimulated with a pool of P. falciparum CSp peptides consisting of C-CSp (PfCS282-383), N-CSp (PfCS22-110) and IDE-CSp (PfCS-NYDNAGTNL).
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