2004 Roster

2004 Women\u27s Softball Roster, George Fox College

Stronger antigen-specific IFN-γ-secreting CD4 T cell responses elicited by SIVgag-LC3b fusion protein compared to SIVgag antigen alone in mice.

<p><b>A</b>, Immunization schedule to evaluate the immunogenicity of the SIVgag-LC3b fusion antigen. C57BL/6 female mice were divided into four groups with 8–10 mice per group. Each mouse was intramuscularly injected 50 μg of the appropriate DNA plasmids at weeks 0 and 2, then boosted intramuscularly with 1×10⁹ vp of corresponding adenoviral vectors at weeks 4 and 6. To assess the immune responses, mice were sacrificed at weeks 4, 6 and 8 after the first immunization to collect splenocytes and serum for analysis of cellular and humoral immune responses. The symbol “↓” represents the time-point of injection; the symbol “<b>Δ</b>” represents the time-point of sacrifice and sample collection. The SIVgag-specific cellular immune responses, as assessed using the IFN-γ ELISPOT assay following stimulation with SIVgag peptide, were shown after DNA-based constructs immunization at week 4 (<b>B</b>) and after adenoviral-based constructs immunization at week 6 (<b>C</b>). The SIVgag-specific cellular immune responses, as assessed using the intracellular IFN-γ cytokine staining assay, were shown after DNA-based constructs immunization at week 4 (<b>D</b>) and after adenoviral-based constructs immunization at week 8 (<b>E</b>). Data were analyzed using the Student’s t-test, and a two-tailed p-value of less than 0.05 was considered statistically significant. These data were expressed as the mean±SEM from four mice samples (*: p<0.05;**: p<0.01; ***: p<0.001). Two independent experiments for the animal immunization were repeated.</p

Stronger SIVgag-specific antibodies elicited in mice using the SIVgag-LC3b fusion protein compared to SIVgag antigen alone.

<p>Mice were immunized and serums were collected at week 8 as shown in the Materials and Methods. The SIV-binding antibody was assessed using ELISA. Data were analyzed using the Student’s t-test, and a two-tailed p-value of less than 0.05 was considered statistically significant (*: p<0.05). Each data point represents the antibody titer from an individual mouse (n = 4). The representative data were obtained from two independent experiments.</p

Construction of DNA vectors and Ad5-based vectors carrying the SIVgag-LC3b gene.

<p><b>A</b>, Schematic representation of constructs carrying various combinations of mouse LC3b gene or SIVgag under the CMV promoter, denoted as LC3b, SIVgag and SIVgag-LC3b, respectively. HeLa cells were transfected with plasmid-based or Ad5-based constructs, and expression of the protein of interest was detected using western blotting analyses. The GAPDH blot demonstrates equal protein loading. <b>B</b>, Expression of the fusion protein using recombinant pVAX plasmid DNA constructs. <b>C</b>, Expression of fusion protein using recombinant Ad5-based constructs. The left blots show proteins detected using anti-SIV Gag and the right blots show proteins detected using the anti-LC3b antibody, respectively.</p

Functionally targeted SIV Gag protein to autophagosomes using confocal microscopy.

<p>HeLa-GFP-LC3 cell line stably expressing GFP-LC3 protein (green fluorescence) were transfected with pVAX-SIVgag plasmid (top) or pVAX-SIVgag-LC3 plasmid (bottom) with or without chloroquine (CQ) treatment, and then stained with anti-SIVgag and Cy3-labeled goat anti-mouse IgG as secondary antibodies (red fluorescence); the nuclei were stained with DAPI (blue fluorescence). The scale bar represents 20 μm. Representative cells are shown from one experiment out of three total experiments.</p

Functionally targeted SIV Gag protein to lysosomes and MHC II compartments using confocal microscopy.

<p><b>A</b>, HeLa cells were transfected with pVAX-SIVgag plasmid (top) or pVAX-SIVgag-LC3 plasmid (bottom) with chloroquine (CQ) treatment, and then stained with mouse anti-SIVgag IgG antibody and rabbit anti-LAMP-2 IgG antibody. Subsequently, Cy3-labeled goat anti-mouse IgG (red fluorescence) and Alexa Fluor 488-labeled goat anti-rabbit IgG (green fluorescence) secondary antibodies were used; the nuclei were stained with DAPI (blue fluorescence). The scale bar represents 20 μm. The data represent three independent experiments. <b>B</b>, RAW 264.7 cells were stimulated with lipopolysaccharide(LPS) and infected with Ad5-SIVgag (top) or Ad5-SIVgag-LC3 plasmid (bottom) with chloroquine (CQ) treatment, and subsequently stained with mouse anti-SIVgag IgG antibody and rat anti-MHC- II IgG antibody. Next, Cy3-labeled goat anti-rat IgG (red fluorescence) and 488-labeled goat anti-mouse IgG (green fluorescence) secondary antibodies were used; the nuclei were stained with DAPI (blue fluorescence). The scale bar represents 20 μm. The data represent three independent experiments. <b>C</b>, HeLa cells were transfected with pVAX, pVAX-SIVgag or pVAX-SIVgag-LC3b for 24 h, and then treated with 75 μM CQ or 200 nM RAPA for 24 h. LC3b-I, LC3b-II and SIVgag were visualized using anti-LC3b and anti-SIVgag immunoblotting, respectively. GAPDH blots demonstrate that CQ or RAPA treatment did not affect the overall protein level. D, SIVgag antigen-specific CD4+ T cells were obtained and isolated using anti-CD4 microbeads, and purified CD4+ T cells (effector cells) were then co-cultured with BMDC cells (target cells). The E:T ratios were 3∶1 or 9∶1. After 48 h, the culture supernatants were measured using the IFN-γ ELISA kit. As a positive control, the BMDC cells stimulated with LPS (1 μg/ml) were used as a positive control, and untreated DMDC cells were used as a negative control. These data were expressed as the mean±SEM from four mice samples.</p