Journal: PLoS ONE

Article Title: Vaccine Platforms Combining Circumsporozoite Protein and Potent Immune Modulators, rEA or EAT-2, Paradoxically Result in Opposing Immune Responses

doi: 10.1371/journal.pone.0024147

Figure Lengend Snippet: CS protein specific immune responses increase in an Ad-CSP dose dependent manner. BALB/cJ mice (n = 3) were injected IM with Ad-CSP ranging from 1×10 7 to 1×10 9 vps/mouse, increasing by half logs. 14 days post injection splenocytes and plasma were collected. (A) ELISpot assays were performed to quantify IFNγ secreting cells from splenocytes stimulated with CS protein peptide, NYDNAGTNL, ex vivo . (B) IFNγ, TNFα, and IL-2 expression by splenocyte derived CD3 + CD8 + T cells was analyzed by flow cytometry following ex vivo stimulation with NYDNAGTNL. (C) Total IgG against CS protein was assessed by ELISA. The bars represent mean ± SD. Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test, *,**,*** denotes significance over naïve, p<0.05, p<0.01, p<0.001.

Article Snippet: Splenocytes were stimulated ex vivo with 4 μg/mL of the >98% pure CS protein immunodominant peptide NYDNAGTNL (amino acids 43–51 of the CS protein sequence) (GenScript Piscataway, NJ) .

Techniques: Injection, Clinical Proteomics, Enzyme-linked Immunospot, Ex Vivo, Expressing, Derivative Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Vaccine Platforms Combining Circumsporozoite Protein and Potent Immune Modulators, rEA or EAT-2, Paradoxically Result in Opposing Immune Responses

doi: 10.1371/journal.pone.0024147

Figure Lengend Snippet: Co-vaccination with Ad-CSP and Ad-EAT2 dramatically increases IFNγ secreting splenocytes in response to stimulation with CS protein epitope, NYDNAGTNL. BALB/cJ mice were injected IM with either 5×10 7 vps/mouse of Ad-CSP and 5×10 7 vps/mouse Ad-GFP or 5×10 7 vps/mouse of Ad-CSP and 5×10 7 vps/mouse of either (A) Ad-GFP/rEA (n = 5) or (B) Ad-EAT2 (n = 6). Splenocytes were collected 14 days post co-injection. ELISpot were performed on the splenocytes of these mice stimulated with NYDNAGTNL peptide to assess the amount of IFNγ secreting cells. The bars represent mean ± SD. Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test,* Denotes significance over naïve animals, p<0.05. Representative figures of two independent experiments.

Article Snippet: Splenocytes were stimulated ex vivo with 4 μg/mL of the >98% pure CS protein immunodominant peptide NYDNAGTNL (amino acids 43–51 of the CS protein sequence) (GenScript Piscataway, NJ) .

Techniques: Injection, Enzyme-linked Immunospot

Journal: PLoS ONE

Article Title: Vaccine Platforms Combining Circumsporozoite Protein and Potent Immune Modulators, rEA or EAT-2, Paradoxically Result in Opposing Immune Responses

doi: 10.1371/journal.pone.0024147

Figure Lengend Snippet: Co-vaccination with Ad-CSP and Ad-EAT2 resulted in increased NYDNAGTNL tetramer positive CD8 + T cells as well as improved IFNγ secretion from CD8 + T cells. BALB/cJ mice (n = 6) were co-injected IM with 5×10 7 vps/mouse of Ad-CSP and 5×10 7 vps/mouse of Ad-EAT2 or 5×10 7 vps/mouse of Ad-CSP and 5×10 7 vps/mouse of Ad-GFP. (A) Peripheral Blood Mononuclear Cells (PBMCs) were stained with CD8-Alexa Flour700, CD3-APC-Cy7, and CSP (NYD)-Tetramer. (B–C) Intracellular staining was performed on splenocytes after stimulation with NYDNAGTNL peptide. Cells were stained with CD8-Alexa Flour700, CD3-APC-Cy7, ViViD, IFNg-APC, and Perforin-PE antibodies. The bars represent mean ± SD. Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test, *, **, *** denotes significance over naïve animals, p<0. 05, p<0. 01, p<0.001.

Article Snippet: Splenocytes were stimulated ex vivo with 4 μg/mL of the >98% pure CS protein immunodominant peptide NYDNAGTNL (amino acids 43–51 of the CS protein sequence) (GenScript Piscataway, NJ) .

Techniques: Injection, Staining

Journal: PLoS ONE

Article Title: Vaccine Platforms Combining Circumsporozoite Protein and Potent Immune Modulators, rEA or EAT-2, Paradoxically Result in Opposing Immune Responses

doi: 10.1371/journal.pone.0024147

Figure Lengend Snippet: Co-vaccination of mice with Ad-CSP and Ad-EAT2 increased specific killing cells pulsed with CS protein peptides. BALB/cJ mice (n = 4) were co-injected IM with either 1×10 8 vps/mouse Ad-CSP and 1×10 8 vps/mouse Ad-GFP or 1×10 8 vps/mouse Ad-CSP and 1×10 8 vps/mouse Ad-EAT2 on Day 0. Day 14 splenocytes were collected from naïve mice and pulsed with either NYDNAGTNL peptide or an irrelevant peptide. NYDNAGTNL pulsed splenocytes were stained with a high concentration of CFSE while splenocytes pulsed with irrelevant peptide were stained with a low concentration of CFSE. Stained splenocytes were then combined in equivalent doses. 8 million cells were then injected IV into naïve, Ad-CSP+Ad-GFP co-vaccinated, or Ad-CSP+Ad-EAT2 co-vaccinated mice. After 18 hrs splenocytes from these mice were collected and analyzed by flow cytometry to assess the amount of NYDNAGTNL specific killing. % Specific killing = 1-((%CFSE High /%CFSE Low ) immunized /(%CFSE High /CFSE Low ) non-immunized ). * denotes significant difference between treatments p<0.05.

Article Snippet: Splenocytes were stimulated ex vivo with 4 μg/mL of the >98% pure CS protein immunodominant peptide NYDNAGTNL (amino acids 43–51 of the CS protein sequence) (GenScript Piscataway, NJ) .

Techniques: Injection, Staining, Concentration Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Decoding the absolute stoichiometric composition and structural plasticity of α-carboxysomes

doi: 10.1101/2021.12.06.471529

Figure Lengend Snippet: ( A ) QconCAT DNA fragment was designed from selected gene sequences from the H. neapolitanus operon that expresses α-carboxysome proteins. The stable isotopes ([ 13 C 6 , 15 N 4 ] arginine and [ 13 C 6 , 15 N 2 ] lysine) labelled QconCAT peptide fusion was expressed via a cell-free system, purified and quantified and added to four replicates samples of isolated native/recombinant α-carboxysomes from H. neapolitanus and E. coli . The absolute quantity and stoichiometry of unlabelled signature peptides for carboxysomal proteins were calculated by LC-MS analysis. One signature peptide for CbbQ, LLVKAGK was shown here as an example. ( B ) SDS-PAGE of isolated native/recombinant α-carboxysomes showing the majority bands of α-carboxysome proteins. ( C ) EM images of isolated native/recombinant α-carboxysomes.

Article Snippet: The data for label-free quantification search against H. neapolitanus protein database are available at: https://simplifi.protifi.com/#/p/e527f900-3e50-11eb-808d-871337eb317b .

Techniques: Purification, Isolation, Recombinant, Liquid Chromatography with Mass Spectroscopy, SDS Page

Journal: bioRxiv

Article Title: Decoding the absolute stoichiometric composition and structural plasticity of α-carboxysomes

doi: 10.1101/2021.12.06.471529

Figure Lengend Snippet: ( A ) Protein index of all recognized proteins in label-free quantification. In native carboxysome samples, McdAB-like proteins are both detected. The full protein list provided in Supplemental File; ( B ) Comparison of QconCAT and label-free quantification. Quantification was normalized to equal total protein quantity. Proteins with the abundance difference greater than 30% from average are labelled in red; (C) Quantification of all QconCAT candidate peptides for H. neapolitanus carboxysomes. Overall good agreements are found within candidate peptides for same protein with the exception of peptide 3 of CsoSCA (34 ± 4 % lower than the other two) and CbbO (21 ± 3 %) in native carboxysome samples.

Article Snippet: The data for label-free quantification search against H. neapolitanus protein database are available at: https://simplifi.protifi.com/#/p/e527f900-3e50-11eb-808d-871337eb317b .

Techniques: Quantitative Proteomics, Comparison