Journal:

Article Title: Induction of Specific T-Cell Responses, Opsonizing Antibodies, and Protection against Plasmodium chabaudi adami Infection in Mice Vaccinated with Genomic Expression Libraries Expressed in Targeted and Secretory DNA Vectors

doi: 10.1128/IAI.71.8.4506-4515.2003

Figure Lengend Snippet: In vitro proliferation of splenocytes from individual BALB/c mice vaccinated i.d. with the gene gun. Mice were vaccinated with VR1020/30K, MCP-3/30K, and CTLA4/30K three times at 2-week intervals. Splenocytes were harvested 10 days after the final vaccination. Following 72 h of stimulation with 2 × 106 IRBCs or 2 × 106 RBCs, [3H]thymidine was added for 18 h. Numbers of counts per minute of radioactivity incorporated by cells were then recorded. Splenocytes vaccinated with empty vector control DNA (VR1020, MCP-3, or CTLA4) were also stimulated with 2 × 106 RBCs, but no incorporation of [3H]thymidine was observed (data not shown). Splenocytes from all individual splenocyte cultures responded to concanavalin A stimulation (data not shown). The mean number of counts per minute (bars) is shown. ∗, P < 0.01; ∗∗, P < 0.05. The insert shows the response of splenocytes from hyperimmune control mice (mice that had survived P. chabaudi adami infection) that were used as a positive control for IRBCs.

Article Snippet: The MCP-3 PCR fragment was digested with Not I and Bam HI and inserted into the VR1012 vector (VICAL) digested with the same enzymes to produce the MCP-3 vector ( 29 ).

Techniques: In Vitro, Radioactivity, Plasmid Preparation, Infection, Positive Control

Journal:

Article Title: Induction of Specific T-Cell Responses, Opsonizing Antibodies, and Protection against Plasmodium chabaudi adami Infection in Mice Vaccinated with Genomic Expression Libraries Expressed in Targeted and Secretory DNA Vectors

doi: 10.1128/IAI.71.8.4506-4515.2003

Figure Lengend Snippet: IFN-γ (A) and IL-4 (B) production by splenocytes from BALB/c mice vaccinated with the VR1020/30K, MCP-3/30K, and CTLA4/30K libraries. Splenocytes from individual mice were cultured as described in the legend to Fig. ​Fig.1.1. After 72 h of stimulation with 2 × 106 IRBCs or 2 × 106 RBCs, supernatants were harvested and analyzed by ELISA for the presence of IFN-γ and IL-4. Splenocytes from mice vaccinated with empty vector control DNA (VR1020, MCP-3, or CTLA4) were also stimulated with 2 × 106 RBCs, but IFN-γ and IL-4 responses were not observed (data not shown). Unstimulated splenocytes did not produce detectable IFN-γ or IL-4. The mean level of IFN-γ or IL-4 production is shown (bars). The raw data were log10 transformed and compared by using the Student t test. ∗, P < 0.05.

Article Snippet: The MCP-3 PCR fragment was digested with Not I and Bam HI and inserted into the VR1012 vector (VICAL) digested with the same enzymes to produce the MCP-3 vector ( 29 ).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Transformation Assay

Journal:

Article Title: Induction of Specific T-Cell Responses, Opsonizing Antibodies, and Protection against Plasmodium chabaudi adami Infection in Mice Vaccinated with Genomic Expression Libraries Expressed in Targeted and Secretory DNA Vectors

doi: 10.1128/IAI.71.8.4506-4515.2003

Figure Lengend Snippet: Phagocytosis of P. chabaudi adami-IRBCs preincubated with sera from mice vaccinated with VR1020/30K, MCP-3/30K, and CTLA4/30K (and control vector DNA). (A) Percentage of macrophages ingesting IRBCs after preincubation with sera. All six groups contained 10 BALB/c mice each. The values are expressed as percentages of macrophages containing IRBCs out of a total of 200 counted. The number of IRBCs contained within an individual macrophage for each vaccine group is also shown. Data are expressed as means ± standard errors of the means. a, P < 0.05; b, P < 0.01; c, P < 0.001; d, P < 0.0001. (B) Percentage of macrophages ingesting IRBCs after incubation with pooled sera from naive (unvaccinated) mice (n = 6) and pooled sera from mice vaccinated with MCP-3/MSP4/5 DNA (n = 6) as a positive control (29). (C) Incubation of macrophages with a monoclonal antibody specific for Fcγ II and Fcγ III receptors inhibits phagocytosis of IRBCs. Macrophages were treated with a α-Fcγ monoclonal antibody, followed by incubation with IRBCs previously exposed to sera from mice vaccinated with each genomic library (30K + α-FCR mAB), or they were left untreated and incubated with IRBCs exposed to sera from vaccinated mice as a positive control (30K). The data shown are expressed as means ± standard errors of the means of the percentages of macrophages ingesting IRBCs. Five serum samples from mice vaccinated with each of the VR1020/30K, MCP-3/30K, and CTLA4/30K libraries were tested, giving a total of 15 individual samples. Treated (30K + α-FCR mAB) and untreated (30K) groups were compared by using the Student t test.

Article Snippet: The MCP-3 PCR fragment was digested with Not I and Bam HI and inserted into the VR1012 vector (VICAL) digested with the same enzymes to produce the MCP-3 vector ( 29 ).

Techniques: Plasmid Preparation, Incubation, Positive Control

Journal:

Article Title: Induction of Specific T-Cell Responses, Opsonizing Antibodies, and Protection against Plasmodium chabaudi adami Infection in Mice Vaccinated with Genomic Expression Libraries Expressed in Targeted and Secretory DNA Vectors

doi: 10.1128/IAI.71.8.4506-4515.2003

Figure Lengend Snippet: Survival curves of mice vaccinated with VR1020/30K, MCP-3/30K, and CTLA4/30K genomic libraries and challenged with virulent P. chabaudi adami. Mice were vaccinated three times at 2-week intervals via the gene gun, followed by challenge with 100,000 P. chabaudi adami-IRBCs 2 weeks later. (A) Evaluation of efficacies of CTLA4/30K and VR1020/30K libraries. (B) Evaluation of efficacy of MCP-3/30K library. (C) Comparative evaluations of all three genomic libraries. #, P = 0.05; ∗, P < 0.05 compared to empty control vectors (Mantel-Haenszel test).

Article Snippet: The MCP-3 PCR fragment was digested with Not I and Bam HI and inserted into the VR1012 vector (VICAL) digested with the same enzymes to produce the MCP-3 vector ( 29 ).

Techniques: