Using more sophisticated flow cytometry-based assay techniques the balance and frequency of the cellular immune response is further elucidated. C and D) and the Gag peptide response (panels E and F). Panels A, C and E show the intra-muscular vaccination responses, while panels B, D and F show the intra-dermal vaccination responses. The y-axis represents the magnitude of the response (IFN SFC/106 PBMC), while the time post-vaccination initiation (days) is shown on the x-axis. Data is presented as corrected values (test article – background). The dotted line (27 SFC/106 PBMC) represents the limit of detection for the validated ELISPOT assay and the red arrows denote the timing of the vaccination series.(0.10 MB PDF) pone.0013983.s002.pdf (95K) GUID:?B0F5F852-8E6E-4377-AD35-243EC256CAF9 Figure S3: Gating strategy applied to the multifunctional flow cytometric analyses. A representative sample is shown for the gating strategy used for viable, CD3+ lymphocyte identification and subsequent subdivision into CD4+ and CD8+ T cells (panel A). A functional positive response is shown for both CD4+ T cells (IL-2 and TNF) and CD8+ T cells (CD107a and MIP-1) in response to the CM235 Env peptide pool (panel B).(0.22 MB PDF) pone.0013983.s003.pdf (213K) GUID:?9232FE0F-2EC8-4684-AE6E-0817895BE008 Protocol S1: Trial Protocol.(0.75 MB PDF) pone.0013983.s004.pdf (735K) GUID:?7492F477-47FB-493A-A194-1B9E7B8DEAD7 Checklist S1: CONSORT Checklist.(0.23 MB DOC) pone.0013983.s005.doc (220K) GUID:?3A2585A1-30EE-4DCA-A6A0-E4C9E3A32A55 Abstract Background We conducted a Phase I randomized, dose-escalation, route-comparison trial of MVA-CMDR, a candidate HIV-1 vaccine based on a recombinant modified vaccinia Ankara viral vector expressing HIV-1 genes inserts derived from a CRF01_AE HIV-1 isolate from Chiang Mai, Thailand, referred to here as MVA-CMDR (Chiang Mai Double Recombinant). The construction details and pre-clinical testing of this vaccine were published earlier [8]. MVA-CMDR has been used as a RU-SKI 43 heterologous boost at the Karolinska Institute, Stockholm, Sweden and the Muhumbili University, Dar es Salaam, Tanzania in previous multigenic and multiclade DNA-prime/MVA-boost trials [9]. This phase I trial was conducted in the US and Thailand focusing on dose escalation and route comparisons of MVA-CMDR alone for the induction of cellular and humoral immunogenicity in a vaccinia na?ve population. Materials and Methods Study vaccine candidate The MVA-CMDR was developed through collaboration between the Laboratory of Viral Diseases (LVD)/National Institute of Allergy and Infectious Diseases (NIAID) and the Walter Reed Army Instiutite of Research (WRAIR)/US Military HIV Research Program (MHRP) [8]. This multigenic vaccine contains inserts derived from CRF01_AE isolates from Chiang Mai (CM), Thailand (HIV-1 CM235 expressed vaccine antigens may lead to false-positive HIV EIA and/or WB results and thus unblind staff to a volunteer’s allocation (placebo or vaccine). RU-SKI 43 CAPN2 Pre- and post-test HIV counseling was performed at each visit. Vaccinia exposure testing Pre-vaccination sera were sent to V-Bio (St. Louis, MO) for Vaccinia ELISA testing. The enzyme-linked immunosorbent assay (ELISA) for vaccinia measures the level of vaccinia specific antibody (IgG) in serum samples. The immunoenzymatic method allows quantification of the virus specific antibody based on a capture RU-SKI 43 technique and subsequent color development measurement by a spectrophotometer. The vaccinia IgG ELISA procedure has been described previously and was modified as described [11]. Briefly, plates were coated with vaccinia antigen or negative (mock-infected) cell culture lysate. Serial 2-fold dilutions of sera were placed on both antigen-coated and mock-antigen coated wells and incubated for two-hours at 37C. After washing horseradish peroxidase-conjugated anti-human IgG was added to the plate followed by a two hour incubation at 37C. After the incubation period, the plates were washed and ABTS substrate (Kirkegaard and Perry, Gaithersburg, MD) was added. Following a 30-minute incubation at room temperature, stopping solution (1%SDS) was added to the plates and the plates were read at 405/492nm dual wavelength. Linear regression plots were prepared and endpoint titers were determined based on an optical density (OD) cut-off of 0.30 using UnitWin software. Cellular Immunogenicity Assessment Blood Collection Peripheral blood mononuclear cells (PBMC) for cellular immunogenicity assays were isolated from whole blood collected in acid-citrate dextrose anti-coagulant using standard procedures [12]. PBMC were either used fresh or cryopreserved in RPMI media containing 20% fetal calf serum and 10% dimethyl sulphoxide (DMSO) in the vapor phase of liquid nitrogen or electric freezers (Revco) at ?130C. All PBMC processing was undertaken within 6 hours of blood collection, and post-thaw.