lav-blot-1  (Bio-Rad)

Journal: Virology Journal

Article Title: Oral vaccination with a recombinant Salmonella vaccine vector provokes systemic HIV-1 subtype C Gag-specific CD4+ Th1 and Th2 cell immune responses in mice

doi: 10.1186/1743-422X-6-87

Figure Lengend Snippet: The HIV-1 subtype C Gag expression plasmid (pGEM+Gag) and Gag expression by Salmonella . (A) The HIV Gag expression cassette contained the gag gene fused in-frame with the β-galactosidase α-gene and expression was under the E. coli lac ( lactose ) promoter. The plasmid contained an E. coli origin of replication (ori) and ampicillin resistance gene (AmpR). (B) The relative expression of the Gag by the recombinant Salmonella vaccine (aroC+Gag) was determined by SDS-PAGE and (C) the Roche Elecsys ® HIV p24 Ag assay. In the Roche Elecsys ® HIV p24 Ag assay, total bacterial protein lysate was diluted 1/1000 in water and the cut-off index was calculated by the Elecsys ® 2010 analyzer using readings from the negative and positive calibrators.

Article Snippet: HIV-1 Gag-specific IgG responses were confirmed using the New LAV Blot I HIV-1 Western blotting kit (Biorad).

Techniques: Expressing, Plasmid Preparation, Recombinant, SDS Page

Journal: Virology Journal

Article Title: Oral vaccination with a recombinant Salmonella vaccine vector provokes systemic HIV-1 subtype C Gag-specific CD4+ Th1 and Th2 cell immune responses in mice

doi: 10.1186/1743-422X-6-87

Figure Lengend Snippet: HIV-1 subtype C Gag-specific CD4+ Th1 and Th2 cell responses as evaluated by IFN-γ and IL-4 ELISPOT assays . Groups of mice (5 mice per group) were vaccinated three times (day 0, 28 and 56) with live recombinant Salmonella vaccine that expressed HIV-1 Subtype C Gag (aroC+Gag) or an antigen-negative Salmonella control vaccine (aroC+pGEM). Mice were sacrificed on day 84, and splenocytes were prepared from isolated spleens pooled from 5 mice per group then used in IFN-γ (A) and IL-4 (B) ELISPOT assays with R10 medium only (negative assay control) or with the Gag CD4 peptide. Bars are the mean number of spots from triplicate wells and are expressed as SFU/10e6 splenocytes. Differences in immune responses between vaccine groups were analyzed by a two-sample student's t-test. A p < 0.05 was considered statistically significant. The differences in response between stimulated cells and unstimulated cells within the same vaccine group were also analyzed by a student's t-test and a p < 0.05 was considered statistically significant. A p > 0.05 was considered not statistically significant (NS).

Article Snippet: HIV-1 Gag-specific IgG responses were confirmed using the New LAV Blot I HIV-1 Western blotting kit (Biorad).

Techniques: Enzyme-linked Immunospot, Recombinant, Isolation

Journal: Virology Journal

Article Title: Oral vaccination with a recombinant Salmonella vaccine vector provokes systemic HIV-1 subtype C Gag-specific CD4+ Th1 and Th2 cell immune responses in mice

doi: 10.1186/1743-422X-6-87

Figure Lengend Snippet: HIV-1 subtype C Gag-specific CD4+ Th1 and Th2 cell responses as evaluated by the amount of cytokines secreted by stimulated cells . Groups of mice (5 per group) were vaccinated with live recombinant Salmonella vaccine that expressed HIV-1 Subtype C Gag (aroC+Gag) or antigen-negative Salmonella control vaccine (aroC+pGEM) as indicated in Figure 2. Splenocytes isolated and pooled from 5 mice per group on day 84 were incubated in R10 medium only (negative assay control), or stimulated with a Gag CD4 peptide for 48 hrs. Cytokines released into the supernatant were quantified using a mouse Th1/Th2 cytokine bead array (CBA) assay. (A) IFN-γ, (B) TNF-α, (C) IL-4 and (D) IL-5. Each bar in the graphs represents the average picogram amount of cytokine produced per 10e6 splenocytes in 48 hours of stimulation with media or Gag CD4 peptide, for triplicate responses. A p < 0.05 was considered statistically significant. A p > 0.05 was considered not statistically significant (NS).

Article Snippet: HIV-1 Gag-specific IgG responses were confirmed using the New LAV Blot I HIV-1 Western blotting kit (Biorad).

Techniques: Recombinant, Isolation, Incubation, Produced

Journal: Virology Journal

Article Title: Oral vaccination with a recombinant Salmonella vaccine vector provokes systemic HIV-1 subtype C Gag-specific CD4+ Th1 and Th2 cell immune responses in mice

doi: 10.1186/1743-422X-6-87

Figure Lengend Snippet: HIV-1 subtype C Gag-specific serum IgG responses in mice vaccinated with recombinant Salmonella vaccine vector . Groups of mice (5 per group) were vaccinated with live recombinant Salmonella vaccine that expressed HIV-1 Subtype C Gag (aroC+Gag) or an antigen-negative Salmonella control vaccine (aroC+pGEM) as indicated in Figure 2. Serum (pooled from 5 mice per group) was isolated from blood taken before each vaccination on day 0, 28 and 56 and just before sacrifice on day 84. (A) The HIV-1 Gag-specific IgG for each group of mice with a 1/100 serum dilution. The data are the ratio of the OD 405 nm for vaccinated mice and the OD 405 nm for the day 0 serum (pre-bleed). (B) The HIV-1 Gag-specific IgG1 and IgG2a were measured in serum of each group of mice on Day 84 with a 1/100 serum dilution. Each bar represents the mean OD 405 nm value. Differences in antibody responses between vaccine groups at different time points were analyzed by a two-sample student's t-test and a p < 0.05 was considered statistically significant. A p > 0.05 was considered not statistically significant (NS).

Article Snippet: HIV-1 Gag-specific IgG responses were confirmed using the New LAV Blot I HIV-1 Western blotting kit (Biorad).

Techniques: Recombinant, Plasmid Preparation, Isolation