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:

Journal: Molecular Plant Pathology

Article Title: The influence of cis ‐acting P 1 protein and translational elements on the expression of P otato virus Y helper‐component proteinase ( HCP ro) in heterologous systems and its suppression of silencing activity

doi: 10.1111/mpp.12025

Figure Lengend Snippet: Quantification of the accumulation of helper‐component proteinase (HCPro) expressed from construct P1‐6x‐HCPro and from construct 6x‐HCPro and of the steady‐state levels of their mRNAs in the presence or absence of heterologous suppressors of silencing in agroinfiltrated patches of Nicotiana benthamiana leaves, in two independent experiments (left and right sets of panels, respectively). In each experiment, patches were infiltrated with empty vector (first lane), construct P1‐6x‐HCPro plus empty vector (second lane) and construct 6x‐HCPro plus vectors expressing: P1, empty vector, the Cucumber mosaic virus 2b protein suppressor of silencing and (in the second experiment only) the Tomato bushy stunt P19 suppressor of silencing (third, fourth and fifth lanes, respectively). Analyses of infiltrated tissue were made at 4 days post‐agroinfiltration. HCPro mRNA accumulation in infiltrated tissues was visualized by Northern blot (top panel), and also quantified by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) (middle panel). Protein accumulation was visualized by Western blot (bottom panel). The panels below the Northern blots show the total nucleic acid samples stained with ethidium bromide in separate gels, as controls of equal loading. The panels below the Western blots show the Ponceau S‐stained membranes after blotting, as controls of loading.

Article Snippet: The ligation product was used to amplify a PCR fusion fragment using the P1 5' oligonucleotide and the 3' oligonucleotide at the HCPro encoding sequence (nucleotides 319–339).

Techniques: Construct, Plasmid Preparation, Expressing, Virus, Northern Blot, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Staining

Journal: Molecular Plant Pathology

Article Title: The influence of cis ‐acting P 1 protein and translational elements on the expression of P otato virus Y helper‐component proteinase ( HCP ro) in heterologous systems and its suppression of silencing activity

doi: 10.1111/mpp.12025

Figure Lengend Snippet: The effects of alterations in the Kozak context on helper‐component proteinase (HCPro) translatability, accumulation and activity. An alanine was introduced between the starting methionine and the 6 × histidine tag encoded by construct 6x‐HCPro [Met‐Ala‐6xHis; construct 6x‐HCPro (Ala)] to assess whether an improved Kozak context sequence (from ATCCATGCA to ATCCATGGC) would increase the transient accumulation of HCPro and its suppressor activity. (A) Quantification of the steady‐state accumulation of protein expressed from construct 6x‐HCPro (Ala) vs. that from either construct 6x‐HCPro or P1‐6x‐HCPro in agroinfiltrated patches (top Western blot panel), and of their corresponding mRNAs [bottom quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) chart] Patches were infiltrated with empty vector (first lane), construct P1‐6x‐HCPro (second and third lanes), construct 6x‐HCPro (fourth and fifth lanes) and construct P1‐6x‐HCPro (Ala) (sixth to ninth lanes), together with either empty vector (lanes labelled ‘–’) or a binary vector expressing P19 (lanes labelled ‘+’). Each lane corresponds to a different patch. Western blot analyses of infiltrated tissue were made at 4 days post‐agroinfiltration. A densitometric analysis of the HCPro bands is shown below the Western blot. Data are given as a percentage of protein relative to that found in the patch in which construct P1‐6x‐HCPro plus the empty vector were co‐infiltrated (100%). The addition of alanine increased the levels of suppressor protein, from undetectable to over 30% of that found in the patch co‐infiltrated with construct P1‐6x‐HCPro plus empty vector. (B) Quantification of the ability of the same constructs to suppress the silencing of a co‐infiltrated green fluorescent protein (GFP) reporter. A densitometric analysis of the reporter bands is shown below the Western blot. Data are given as a percentage of protein detected relative to that found in the patch in which the GFP reporter and construct P1‐6x‐HCPro were co‐infiltrated (a value of 100%). The extra alanine increased the accumulation of the GFP reporter in the infiltrated patches to 56%–63% of that found in the patch co‐infiltrated with construct P1‐6x‐HCPro plus empty vector. In both (A) and (B), lane M shows molecular weight markers, and the small panels below the Western blots show the membranes stained with Ponceau‐S as controls of loading.

Article Snippet: The ligation product was used to amplify a PCR fusion fragment using the P1 5' oligonucleotide and the 3' oligonucleotide at the HCPro encoding sequence (nucleotides 319–339).

Techniques: Activity Assay, Construct, Sequencing, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Plasmid Preparation, Expressing, Molecular Weight, Staining