Expression and trafficking of mouse and human LAMP/<i>gag.</i>

<p>(A) Western blot analysis of human 293 cells transfected with mLAMP/<i>gag</i> and hLAMP/<i>gag</i> plasmids. Samples were probed with an anti-Gag monoclonal antibody. The molecular weight markers are indicated on the right. (B) DCEK cells were transfected with the hLAMP/<i>gag</i> plasmid and stained with anti-Gag (red) or anti-MHC II (green) monoclonal antibodies. Digitally merged image shows co-localization of the hLAMP/<i>gag</i> chimera- and MHC II-containing compartments (yellow).</p

Immune responses in mice.

<p>Mice were injected i.m. on days 1 and 21 with 50 µg of the indicated pITR plasmids encoding mouse or human LAMP or LAMP/<i>gag</i> chimeras. (A) Humoral responses. Blood samples were collected 7 days after the second immunization. Each titration curve indicates the OD₄₅₀ of Gag-specific total IgG present in serial dilutions of pooled serum from individual groups of mice. (B) CD4⁺ immune responses. CD4⁺ IFN-γ⁺ secretion from splenocytes of mice injected twice with the indicated plasmids. Animals were sacrificed on day 28, and splenocytes were prepared for assay of CD4⁺ T-cell responses and incubated in the presence or absence of antigen. A representative experiment is shown. Data are Mean±SD after subtraction of the background control values. (C) CD8⁺ immune responses. On day 21, mice were injected i.p. with 10⁷ PFU of rVV-<i>gag-pol</i>. Five days later, 10 µg of the H-2Kd-binding HIV-1 Gag peptide was injected i.v., and splenocytes were harvested after 2 h and analyzed <i>ex vivo</i>. Gag-specific CD8⁺ T-lymphocytes producing IFN-γ was quantified by flow cytometry and tetramer binding in the CD8⁺ splenic population. Cytolytic activity was also analyzed in a 4-h ⁵¹Cr release assay using P815 target cells pulsed with the H-2Kd-binding HIV-1 Gag peptide. The effector cells from the various groups of immunized mice were used as indicated. Nonspecific lysis (using unpulsed P815 target cells) was <5% for all groups (not shown). A representative experiment is shown.</p

IgA and IL-6 immune responses of hLAMP/<i>gag</i> immunized macaques.

<p>Gag-specific mucosal immune responses of immunized Rhesus macaques were quantitated by ELISA for IgA and IL-6. (A) Serum IgA levels of the individual macaques at different time-points. (B) IL-6 production of PBMCs from individual macaques stimulated at week 32 with recombinant Gag protein after four DNA immunizations. (C) IgA levels of external secretions of individual macaques.</p

Humoral immune responses of immunized macaques.

<p>Gag-specific antibody responses of Rhesus macaques immunized with hLAMP/<i>gag</i> plasmid were measured by ELISA. (A) Sera diluted 1∶100 from individual macaques was assayed for total anti-Gag antibodies (IgG). (B) End-point titers were measured at week 24 after four DNA immunizations. The reported titers correspond to the reciprocal of the highest serum dilution that gave an OD value three times higher than that of the corresponding dilution of a non-immune serum.</p

T-cell immune response of immunized macaques.

<p>IFN-γ ELISPOT assays were performed with PBMC samples from each of five Rhesus macaques immunized with 5 mg of hLAMP/<i>gag</i> DNA plasmid at weeks 0, 4, 14, 22 and 38. Each time point represents the average number of cells activated in separate assays by rGag protein or Gag 15- or 20-aa peptides, performed at two laboratories in duplicate or triplicate. Each bar is the average number of cells activated in 8 to 20 ELISPOT wells+/−S.D. and represents the number of IFN-γ⁺ cells per 10⁶ PBMCs.</p

Comparison of the T-cell responses of immunized macaques to Gag 15- and 20-aa peptide pools.

<p>Comparison of the average number of IFN-γ⁺ cells activated by Gag 15- and 20-aa peptide pools in the ELISPOT assays, using PBMCs from five individual Rhesus macaques after three DNA immunizations. Each bar represents the number of IFN-γ⁺ cells per 10⁶ PBMCs.</p

Protocol for immunization of Rhesus macaques with the hLAMP/<i>gag</i> plasmid.

<p>Immunization and blood sample collection from five Rhesus macaques immunized five times with 5 mg of hLAMP/<i>gag</i> DNA plasmid were carried out as shown.</p

T-cell immune responses to twelve pools of ten 15-aa Gag peptides each.

<p>T-cell responses of PBMCs from immunized Rhesus macaques to 12 pools, each consisting of ten gag 15-aa peptides overlapping by 11-aa, were measured by use of ELISPOT assays at week 20 after three DNA immunizations. Each bar represents the frequency of IFN-γ⁺ cells per 10⁶ PBMCs.</p

CD4⁺ T-cell immune responses of immunized macaques.

<p>ELISPOT assays show the average number of IFN-γ⁺ cells activated by recombinant Gag protein with CD8⁺-depleted PBMCs from five individual rhesus macaques after four DNA immunizations (at week 36). Each bar is the average count from duplicate wells+/−S.D. and represents the number of IFN-γ⁺ cells per 10⁶ CD8⁺-depleted PBMCs.</p