codon-optimized cdna fragments  (Ginkgo Bioworks)

Journal: PLoS ONE

Article Title: The Immunodominance Change and Protection of CD4 + T-Cell Responses Elicited by an Envelope Protein Domain III-Based Tetravalent Dengue Vaccine in Mice

doi: 10.1371/journal.pone.0145717

Figure Lengend Snippet: (A) Schematic diagram of two ED3-expressing plasmids, pDV13-ED3 and pDV24-ED3, generated by cloning DNA fragments consisting of tandem repeats of ED3 from DENV-1 and 3, and DENV-2 and 4 into the pVax-1 vector between the Xho I and Apa I sites. The positions of the immunoglobulin light chain signal peptide (Ig) or three repeats of GGGGS linker (linker) are also indicated. (B) The presence of bivalent ED3 in the culture supernatants or cell lysates of 293Trex cells transfected with pVax-1, pDV13-ED3 or pDV24-ED3 for 24 h was detected with an ED3-specific monoclonal antibody. The expression of β-actin cellular protein in the cell lysate was used as a control. The recombinant ED3 proteins from DENV-1 to 4 were expressed in E . coli and purified with an affinity column. The final products were analyzed by SDS-PAGE (C) or Western blotting (D) with hyperimmune sera.

Article Snippet: The codon-optimized cDNA fragments encoding bivalent ED3 were synthesized (Genscript, Piscataway, NJ, USA) and inserted into the pVax-1 expression vector between Xho I and Apa I sites.

Techniques: Expressing, Generated, Cloning, Plasmid Preparation, Transfection, Control, Recombinant, Purification, Affinity Column, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: The Immunodominance Change and Protection of CD4 + T-Cell Responses Elicited by an Envelope Protein Domain III-Based Tetravalent Dengue Vaccine in Mice

doi: 10.1371/journal.pone.0145717

Figure Lengend Snippet: Groups of 6- to 8-week-old BALB/c female mice (n = 4) were immunized three times at 2-week intervals with pVax-1, pTDV-ED3 DNA vaccine (pTDV+pTDV), rTED3 protein vaccine (rTED3+rTED3) or prime-boost (pTDV+rTED3) as indicated in the top of figure. One week after last immunization, spleen cells were harvested for the detection of IFN-γ (A) or IL-4 (B) production specific to the ED3 of each serotype by ELISPOT assay. The results are presented with the mean and standard deviation (SD) of spot-forming cells (SFC) per million splenocytes. The significance shown in the graph was determined in comparison to the pVax-1 group, if there is no other indication. The typical results from one out of two independent experiments are presented.

Article Snippet: The codon-optimized cDNA fragments encoding bivalent ED3 were synthesized (Genscript, Piscataway, NJ, USA) and inserted into the pVax-1 expression vector between Xho I and Apa I sites.

Techniques: Enzyme-linked Immunospot, Standard Deviation, Comparison

Journal: PLoS ONE

Article Title: The Immunodominance Change and Protection of CD4 + T-Cell Responses Elicited by an Envelope Protein Domain III-Based Tetravalent Dengue Vaccine in Mice

doi: 10.1371/journal.pone.0145717

Figure Lengend Snippet: Groups of 6- to 8-week-old BALB/c female mice (n = 6) were immunized three times at 2-week intervals with pVax-1, pTDV-ED3, rTED3 or prime-boost as the same schedule in . The results showing here come from the sera sample collected at week 6 after immunization. (A) The ED3-specific IgG titers assayed by ELISA is presented with the mean and SD. (B) The neutralizing antibody titers against four serotypes of DENV were determined by FRNT, and the endpoint titer leading to ≥50% reduction (FRNT 50 ) is shown. Mann-Whitney t-tests were used for statistical analyses, and the significance compared to the pVax-1 group is shown, if nothing else is indicated. The data are representative of two independent experiments with similar results.

Article Snippet: The codon-optimized cDNA fragments encoding bivalent ED3 were synthesized (Genscript, Piscataway, NJ, USA) and inserted into the pVax-1 expression vector between Xho I and Apa I sites.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: PLoS ONE

Article Title: The Immunodominance Change and Protection of CD4 + T-Cell Responses Elicited by an Envelope Protein Domain III-Based Tetravalent Dengue Vaccine in Mice

doi: 10.1371/journal.pone.0145717

Figure Lengend Snippet: Mice (n = 4 per group) were immunized with either pDV13-ED3 or pDV24-ED3 and boosted with recombinant DENV-4 ED3 (rD4) with alum 2 weeks later. (A) Spleen cells were harvested 1 week after the boost, and T-cell epitopes were mapped using IFN-γ ELISPOT. The mean and SD of IFN-γ SFC per million cells responding to sixteen individual peptides for each serotype ED3 are shown. The significance shown in the graph was determined in comparison to other individual peptides. (B) Part of spleen cells were treated with anti-CD4 antibody conjugated with magnetic beads (MACs) for CD4 + cells depletion or were left untreated to analyze the CD4 dependence of specific IFN-γ response by ELISPOT. The mean and SD of IFN-γ SFC per million cells with or without CD4-depletion are indicated. (C) The amino acid sequences of ED3 peptide number 10 (E 349-363 ) and 4 (E 313-327 ) from DENV-1 to 4 are listed.

Article Snippet: The codon-optimized cDNA fragments encoding bivalent ED3 were synthesized (Genscript, Piscataway, NJ, USA) and inserted into the pVax-1 expression vector between Xho I and Apa I sites.

Techniques: Recombinant, Enzyme-linked Immunospot, Comparison, Magnetic Beads

Journal: PLoS ONE

Article Title: The Immunodominance Change and Protection of CD4 + T-Cell Responses Elicited by an Envelope Protein Domain III-Based Tetravalent Dengue Vaccine in Mice

doi: 10.1371/journal.pone.0145717

Figure Lengend Snippet: Groups of 6- to 8-week-old BALB/c female mice (n = 4) were immunized three times at 2-week intervals with pTDV-ED3, rTED3 or prime-boost as indicated in . Spleen cells from two mice per group were harvested one week after either the 1 st or 2 nd boost for the detection of IFN-γ production by ELISPOT. The mean and SD of SFC per million splenocytes responding to stimulation with the indicated individual peptides are shown.

Article Snippet: The codon-optimized cDNA fragments encoding bivalent ED3 were synthesized (Genscript, Piscataway, NJ, USA) and inserted into the pVax-1 expression vector between Xho I and Apa I sites.

Techniques: Enzyme-linked Immunospot

Journal: PLoS ONE

Article Title: The Immunodominance Change and Protection of CD4 + T-Cell Responses Elicited by an Envelope Protein Domain III-Based Tetravalent Dengue Vaccine in Mice

doi: 10.1371/journal.pone.0145717

Figure Lengend Snippet: Groups of 6- to 8-week-old BALB/c female mice (n = 6, except n = 4 for pVax-1) were immunized three times at 2-week intervals with pVax-1, pTDV-ED3, rTED3 or prime-boost as the same schedule in . Four weeks after the last immunization, mice were challenged with the intraperitoneal injection of 5 x 10 7 DENV-4 infected K562 cells. (A) Plasma viremia titers from individual mice were determined by viremia assay and are represented as the mean and SD. The significance shown in the graph was determined in comparison to the pVax-1 group, if nothing else is indicated. (B) Spleen cells were harvested for the detection of the ED3-specific IFN-γ production by ELISPOT at 1 month post-challenge. (C and D) The sera collected at 1 month post-challenge were used for the detection of neutralizing titers in an FRNT assay (C) or ED3-specific IgG titers by ELISA (D).

Article Snippet: The codon-optimized cDNA fragments encoding bivalent ED3 were synthesized (Genscript, Piscataway, NJ, USA) and inserted into the pVax-1 expression vector between Xho I and Apa I sites.

Techniques: Injection, Infection, Clinical Proteomics, Comparison, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay