Immunological evaluation of a DNA-Gag^M prime/MVA-Gag^M boost in BALB/c mice.

<p>(<b>A</b>) Vaccination schedule used. (<b>B</b>) Cumulative IFN-γ ELISPOT CD8⁺ and CD4⁺ responses of vaccinated mice to HIV-1 Gag peptides. The ELISPOT assay was done using pooled spleens on the day of sacrifice using three Gag-specific peptides for stimulation. Bars are the mean and standard deviation of the mean responses for the indicated individual peptides from 3 independent experiments. Responses are expressed as sfu/10⁶ splenocytes after background subtraction. Horizontal bars with asterisks indicate statistical significance of the mean responses between the indicated groups. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001; Student t-test of unpaired data. Total frequency of CD8+ (<b>C</b>) and CD4+ (<b>D</b>) T cells producing IFN-γ, IL-2, and/or TNF-α in response to HIV-1 Gag peptide stimulation. ICS and flow cytometry were carried out on pooled spleens per group using three Gag-specific peptides for stimulation. The memory distribution of the cytokine producing T-cells in the central and effector memory compartment (T_CM and T_EM) are represented as pie charts above each corresponding bar per group. Cells were positive for cytokine production if the proportion was ≥ 0.05% after subtracting the background. The cellular phenotype was positive if there were ≥ 10 cells per test.</p

Heterologous prime-boost vaccination with DNA and MVA vaccines, expressing HIV-1 subtype C mosaic Gag virus-like particles, is highly immunogenic in mice - Fig 1

<p><b>p24 Gag production by:</b> (<b>A</b>) <b>DNA vaccines in HEK cells.</b> HEK cells were transfected with 4μg of DNA-Gag^M or DNA-Gag^N and samples taken at the indicated time points. (<b>B</b>) <b>Recombinant MVA in BHK-21 and HeLa cell lines.</b> Permissive (BHK-21) and non-permissive (HeLa) cell lines were infected at an MOI of 0.1 with MVA-Gag^M and samples taken at the indicated time points. Gag p24 was detected using an ELISA assay.</p

Amino acid sequence alignment of HIV-1 subtype C mosaic Gag and strain Du422 Gag.

<p>The P17 (matrix), P24 (capsid), SP-1(spacer region 1), P7 (nucleocapsid), SP-2 (spacer region 2) and P6 domains of Gag are shown. The PTAPP binding site for ESCRT-1 protein, TSG101, is indicated as is the LxxLF motif which interacts with ALIX protein of ESCRT-III. Mouse Gag CD8+ (AMQMLKDTI) and CD4+ (NPPIPVGRIYKRWIILGLNK GagCD4(13) and FRDYVDRFFKTLRAEQATQE GagCD4(17)) epitopes are indicated in black, bold font.</p

Mouse immunization regimen.

<p>Mouse immunization regimen.</p

Recombinant MVA construction.

<p>The transfer vector pTJMVA2 was designed to insert the <i>gag</i>^<i>M</i> gene under the transcriptional control of the mH5 promoter, between the MVA A11R and A12L ORFs. The selection (<i>bsd</i>) and marker (<i>gfp</i>) genes were expressed as a fused protein under the transcriptional control of the pSS MVA synthetic promoter.</p

Schematic representation of the BCG shuttle vectors and the determination of their genetic integrity before and after vaccination.

<p>Schematic representation of (<b>A</b>) pTJBCG3, which was used to make the BCG-Gag^M vaccine, (<b>B</b>) pEM19 which was used to make the BCG^E vaccine. Restriction sites used for cloning and restriction mapping analysis are indicated in black bold type. OriE–<i>E</i>. <i>coli</i> origin of replication; OriM–mycobacterial origin of replication, 19kD ss– 19kD signal sequence; KanR–kanamycin resistance gene. (<b>C</b>) pTJBCG3 digested with <i>Xho</i>I. Lane 1 is a positive control of pTJBCG3 DNA prior to transformation into BCGΔ<i>panCD</i>. Lanes 2–21 contain pTJBCG3 DNA obtained from recombinant BCG-Gag^M vaccine stocks. (<b>D</b>) pEM19 digested with <i>Sma</i>I. Lanes 1–15 contain pEM19 plasmid DNA isolated from recombinant BCG^E vaccine stocks, and lane 16 pEM19 plasmid DNA isolated prior to transformation into BCGΔ<i>panCD</i> (positive control). PCR amplification of DNA from rBCG obtained from the spleens and lymph nodes of mice vaccinated with BCG-Gag^M (Group 2) (<b>E</b>) or BCG^E (Group 5) (<b>F</b>) 11.5 weeks post vaccination. Lane 1 –negative control; Lane 2 –positive control; Lane 3–12 are PCR products from rBCG isolated from homogenised spleen (3–7) or lymph nodes (8–12). Lanes M in <b>C</b>—<b>F</b> contain the molecular weight marker O’GeneRuler^TM 1kb DNA ladder.</p

<i>In vitro</i> expression of Gag by BHK-21 cells infected with MVA-Gag^M.

<p>(<b>A</b>) BHK-21 cells were infected with MVA-Gag^M, wild type MVA (wtMVA), or left uninfected for 72 hours. HIV-1 Gag was detected with anti-Gag antibody ARP432 (α-Gag; top panels) as well as an anti-VACV antibody (α-MVA; bottom panels), followed by an anti-rabbit HRP-conjugated antibody. (<b>B</b>) Cell lysates were prepared from BHK-21 cells infected with MVA-Gag^M (lane 4), wild type MVA (lane 3), or left uninfected (lane 2). BHK cells transfected with a plasmid known to express full length Gag was used as a positive control (lane 1). Western blots were probed with a rabbit anti-HIV-1-p24 Gag antibody (ARP432), followed by an anti-rabbit antibody conjugated to alkaline phosphatase (Sigma-Aldrich, USA). A Precision Plus Protein Kaleidoscope pre-stained standard (lane M; Biorad, USA) was used and the sizes are indicated on the left.</p

Determination of the optimal dosage of MVA-Gag^M to boost a BCG-Gag^M prime.

<p>(<b>A)</b> Mice were primed on day 0 with 2 x 10⁷ cfu BCG-Gag^M (Group 1–3) or BCG^E (Group 4–6) and boosted on day 70 with 10² (Group 1 and 4), 10⁴ (Group 2 and 5), or 10⁶ (Group 3 and 6) pfu MVA-Gag^M. (<b>B</b>) Cumulative IFN-γ ELISPOT CD8⁺ and CD4⁺ responses of vaccinated mice to HIV-1 Gag peptides. The ELISPOT assay was carried out using three Gag-specific peptides for stimulation of pooled splenocytes that were isolated 12 days post the MVA-Gag^M boost. Bars represent the magnitude of net responses to individual peptides, expressed as sfu/10⁶ splenocytes after subtracting the background. (<b>C</b>) and (<b>D</b>) Total frequency of T cells producing IFN-γ, IL-2, and/or TNF-α, after subtracting the background, in response to HIV-1 Gag peptide stimulation following a rBCG prime and an MVA-Gag^M boost at different doses. Cells were positive for cytokine production if the proportion was ≥0.05% after subtracting the background. These results are from a single experiment using pooled splenocytes.</p