<i>In-vivo</i> experimental vaccination regimens.

<p>Five Balb/c mice per group were used to evaluate the humoral immune responses following their injection with T/SA cells infected or co-infected with FP single recombinants. The mice were immunised twice at a 3-week interval. Black triangles, times of mice bleeding; arrows, cell inoculations.</p

Expression of the Gp, Env and CIITA proteins by FP recombinants in CEFs and Vero cells.

<p>The cells were infected by the single or double recombinants and examined by WB to determine Gp (A), Env (B) and CIITA (C) protein expression. Gp and Env proteins were expressed following the infection of either CEFs or Vero cells with the FP<i>gpCIITA</i> or FP<i>envCIITA</i> double recombinants (A, B). The expression of CIITA was high when this was driven by the H6 promoter (C). Co-infection of Vero cells with either the FP<i>gp+</i>FP<i>CIITA</i>_H6 or FP<i>env+</i>FP<i>CIITA</i>_H6 recombinants showed high <i>gp</i> and <i>env</i> expression (D). The Gp and Env proteins were detected using a monkey polyclonal anti-SIV serum, and CIITA using the mouse 7-1H mAb. Proteins were revealed using the ECL system. Cells infected with FPwt were used as the negative controls.</p

CIITA subcellular localisation using confocal microscopy.

<p>The 293T cells were infected with different FP recombinants and analysed using an anti-SIV polyclonal antibody, and anti-HLA-DR and anti-<i>CIITA</i> mAbs. Using anti-SIV serum, the Gp and Env proteins were equally well expressed by FP<i>gp</i> or FP<i>env</i> (C1, E1), as well as by FP<i>gp</i>+FP<i>CIITA</i>_H6 (D1) or FP<i>env</i>+FP<i>CIITA</i>_H6 (F1). After infection with FP<i>CIITA</i>_H6, both HLA-DR and CIITA were expressed (B2, B3). Also, HLA-DR is expressed in cells co-infected with FP<i>gp</i>+FP<i>CIITA</i>_H6 (D2) or FP<i>env</i>+FP<i>CIITA</i>_H6 (F2). As expected, no Gp or Env proteins were present after infection with the FP<i>CIITA</i>_SP or FP<i>CIITA</i>_H6 recombinants (A1, B1), and no HLA-DR or CIITA expression when the cells were infected with FP<i>gp</i> (C2, C3) or FP<i>env</i> (E2, E3). Differential interference contrast (DIC) microscopy of 293T cells is also shown (A5-F5).</p

Analysis of the humoral response.

<p>The anti-Env humoral response was determined using ELISA, with lysed Vero cells previously infected with FP<i>env</i> as the plate-bound antigen. Serum was obtained from all of the mice at T0, T1, T2 and examined. After the second inoculation of T/SA cells, significant increases in the antibody titres were observed in G1, G2 and G3 (T2 <i>vs</i> T1; p <0.001). Also, a significant difference is shown when T/SA cells were infected only with FP<i>env</i> rather than after co-infection with FP<i>env</i>+FP<i>CIITA</i>_H6 (T2, G1 <i>vs</i> G3; p <0.01). The OD₄₅₀ values were expressed after subtracting the T0 values for each mouse. Statistical significances using one-way ANOVA parametric tests and Bonferroni analysis of variance are shown: p <0.01 (**), p <0.001 (***).</p

Promoters inserted into the double or single FP recombinants for the expression of the <i>gp</i>, <i>env</i>, or <i>CIITA</i> transgenes.

<p>Promoters inserted into the double or single FP recombinants for the expression of the <i>gp</i>, <i>env</i>, or <i>CIITA</i> transgenes.</p

CIITA functionality in T/SA cells using FACS analysis.

<p>MHC-II expression was examined after infection of T/SA cells with the FP<i>gp</i>, FP<i>env</i>, FP<i>CIITA</i>_H6, or FP<i>gp</i>+FP<i>CIITA</i>_H6/ FP<i>env</i>+FP<i>CIITA</i>_H6 recombinants. Similar data as those for 293T cells were obtained, which confirmed that MHC-II expression was higher when using FP<i>CIITA</i>_H6 alone (b, e) or in combination with FP<i>gp</i> or FP<i>env</i> (c, f), rather than when the cells were infected with FP<i>gp</i> or FP<i>env</i> alone (a, d). Results are expressed as number of cells <i>vs</i> the intensity of fluorescence in arbitrary units. Histograms represent fluorescence profiles of the cells, incubated with specific anti MHC-II mAb (solid line) or with FITC-conjugated F(ab)2 anti mouse antibody (dashed line).</p

Time-course of the expression of the <i>gp</i>, <i>env</i> and <i>CIITA</i> transcripts in Vero cells using real-time PCR.

<p>After infection with the different recombinants, quantitative expression of the transgenes was evaluated at days 1, 3 and 6 p.i.. Co-infection with the FP<i>gp</i>+FP<i>CIITA</i>_H6 or FP<i>env</i>+FP<i>CIITA</i>_H6 recombinants significantly increased the <i>gp</i> and <i>env</i> expression compared to cells infected with either the FP<i>gp</i> single or FP<i>gpCIITA</i> double recombinants (A, B). Also, CIITA expression was significantly higher when the transgene was driven by the H6 promoter than by the SP promoter (C, I; p <0.001). <i>CIITA</i> expressed by FP<i>CIITA</i> alone either under the SP or H6 promoter is better expressed than when combined with the second <i>gp</i> or <i>env</i> transgene (panel C, Ia <i>vs</i> IIa and IIIa, and Ib <i>vs</i> IIb and IIIb, p <0.001). However, when co-infection with single recombinants is performed, its expression increases over time (panel C, IIb and IIIb, day 6 <i>vs</i> days 1 and 3, p <0.001). Mock-infected cells were always negative, as were cells infected with FPwt (data not shown). The experiment was repeated twice and the results are given as the average of the data. Data were normalised against the RPS7 endogenous housekeeping gene transcript, which indicates N-fold increases <i>vs</i> control mRNA at each time point. Statistical significances using one-way ANOVA parametric tests and Bonferroni analysis of variance are shown: p <0.01 (**), p <0.001 (***).</p

Analysis of CIITA-mediated HLA-DR trasactivation by FACS.

<p>After 293T cell infection, an increase in <i>HLA-DR</i> expression was mainly detected when the cells were infected with FP<i>CIITA</i>_H6 (A, B, b <i>vs</i> a) or co-infected with the FP<i>gp</i>+FP<i>CIITA</i>_H6 or FP<i>env</i>+FP<i>CIITA</i>_H6 single recombinants (A, B, c). HLA-DR expression was enhanced when the CIITA transgene was driven by the H6 promoter (C, b <i>vs</i> a). <i>CIITA</i> better transactivates endogenous HLA-DR after co-infection with FP<i>env</i>+FP<i>CIITA</i>_H6, rather than by the FP<i>envCIITA</i> double recombinant (C, d <i>vs</i> c). HLA-DR expression was observed in cells transiently transfected with pcflag<i>CIITA</i> (about 40% of the cells), as expected (C, e). The experiments were replicated twice with similar results. Results are expressed as number of cells <i>vs</i> the intensity of fluorescence in arbitrary units. Histograms represent fluorescence profiles of the cells, incubated with specific anti HLA-DR mAb (solid line) or with FITC-conjugated F(ab)2 anti mouse antibody (dashed line).</p