Journal: PLoS Pathogens

Article Title: T Cell Inactivation by Poxviral B22 Family Proteins Increases Viral Virulence

doi: 10.1371/journal.ppat.1004123

Figure Lengend Snippet: A) Left Panel : PBMC from VACV-immune subjects (n = 4) were infected with VACV, MPXV Zaire or MPXV US2003 (MOI of 0.5) for 18 h. Poxvirus-specific CD4+ and CD8+ T cell responses were measured by ICCS. Results are normalized to % of VACV-specific response. Right Panel : PBMC from VACV-naïve subjects (n = 3) were infected with indicated viruses or uninfected (UN) and T cells were stimulated with plate-bound αCD3 Ab for 6 h. B) CM9-specific RM CD8+ T-cells were incubated with HFF infected with MPXV US2003 (MOI of 2) in the presence or absence of 10 µM ST246 for 18 h prior to stimulation with CM9-peptide pulsed BLCL cells for 6.5 h. The percentage of IFNγ+TNFα+ cells as measured by ICCS is shown. C) Map of 10 Kb deletions (light grey) or a single ORF 197 deletion (black) in the terminal regions of the MPXV US2003 genome (black). D) Human CD4+ and CD8+ T cell responses to MPXV deletion mutants were determined by ICCS as in A). Infection rates of CD14+ monocytes in PBMC for MPXV US2003, MPXVΔ184-193, MPXVΔ194-197, and MPXVΔ197 were 73%, 81%, 65%, and 70%, respectively. E) Inhibition of CM9-specific CD8+ T-cell stimulation by MPXV US2003 or deletion mutants was measured by ICCS as in B). T cells were co-incubated with HFF cells infected with indicated viruses (MOI of 2, 10 µM ST246) for 18 h and then stimulated with CM9-peptide pulsed BLCLs for 6.5 h.

Article Snippet: Codon-optimized sequences of the C-terminal 3×FLAG (DYKDHDGDYKDHDIDYKDDDDK) fusions of MPXV197 and VARV B22 proteins were synthesized at GenScript (Piscataway, NJ).

Techniques: Infection, Incubation, Inhibition, Cell Stimulation

Journal: PLoS Pathogens

Article Title: T Cell Inactivation by Poxviral B22 Family Proteins Increases Viral Virulence

doi: 10.1371/journal.ppat.1004123

Figure Lengend Snippet: A) Schematic representation of MPXV 197 with predicted signal peptide (SP, blue, SignalP), transmembrane domains (TM, green, TMPred), and N-linked glycosylation sites (red, NetNGlyc 1.0). B) CHO cells were transduced with Ad-197/Ad-tTA (‘Ad-197’) or Ad-tTA only (‘Ad-control’) for 24 hours and lysed in sample buffer prior to electrophoretic separation and immunoblotting with αFLAG. Right panel: Overexposure reveals a >250 kDa band (asterisk). C) CHO cells were transduced as in B). After 24 h, cell surface proteins were biotinylated followed by immunoprecipitation with NeutrAvidin, electrophoretic separation and immunoblotting with αFLAG. D) 24 h after transduction with the indicated expression vectors, CHO cells were metabolically labeled for 45 min followed by chase for 0.5, 1, and 3 h. Cell lysates were immunoprecipitated with αFLAG. In the right panel, samples were treated with EndoH or left untreated prior to electrophoretic separation. EndoH sensitive proteins are indicated by asterisks. E) Sub-cellular localization of C- and N-terminal FLAG fusions of MPXV197 was determined by IFA using αFLAG. CHO cells were either permeabilized (‘Intracellular’) or non-permeabilized (‘Cell-surface’) prior to IFA. Scale bar is 20 µm. Arrows indicate the plasma membrane.

Article Snippet: Codon-optimized sequences of the C-terminal 3×FLAG (DYKDHDGDYKDHDIDYKDDDDK) fusions of MPXV197 and VARV B22 proteins were synthesized at GenScript (Piscataway, NJ).

Techniques: Glycoproteomics, Transduction, Control, Western Blot, Immunoprecipitation, Expressing, Metabolic Labelling, Labeling, Clinical Proteomics, Membrane

Journal: PLoS Pathogens

Article Title: T Cell Inactivation by Poxviral B22 Family Proteins Increases Viral Virulence

doi: 10.1371/journal.ppat.1004123

Figure Lengend Snippet: A) CM9-specific CD8+ T-cells were incubated (18 h) with untreated CHO cells (UN) or CHO cells transduced with either Ad-197/Ad-tTA (‘Ad-197’) or Ad-tTA only (‘Ad-control’) and stimulated with CM9-peptide pulsed BLCLs. The percentage of INFγ+ TNFα+ CD8+ T-cells was determined by ICCS. B) To determine the kinetics of T cell inhibition by MPXV197 CM9-specific T-cells were incubated with Ad-197/Ad-tTA or Ad-tTA-transduced CHO cells for indicated time periods, washed, and stimulated with peptide pulsed BLCLs. C) Human Mtb specific CD8+ T cell clones D466 D6 and D160 1–23 were stimulated with BEAS-2b cells transduced with Ad-197/Ad-tTA or Ad-tTA only in the presence of CFP10 2-12 peptide or pronase digested Mtb cell wall, respectively. For MHC-independent stimulation, both clones were incubated with PHA. The number of IFNγ+ T cells was measured by ELISPOT. D) CM9-specific CD8+ T cells were incubated (18 h) with CHO cells transduced with Ad-197/Ad-tTA or Ad-tTA, washed, and stimulated either with PMA/Iono or CM9-peptide pulsed BLCLs. Left panel : The percentages of INFγ+ TNFα+ T-cells were determined by ICCS with stimulation in the presence of uninfected CHO cells set to 100% (MAX). Right Panel : The percent live CD8+ T cells was determined by LIVE/DEAD Fixable Dead Cell Stain. E) MaMu-A*01/CM9 tetramer staining of CM9-pecific CD8+ T cells after 18 h of incubation with MPXV197-expressing CHO cells (‘Ad-197’) or control cells (‘Ad-control’).

Article Snippet: Codon-optimized sequences of the C-terminal 3×FLAG (DYKDHDGDYKDHDIDYKDDDDK) fusions of MPXV197 and VARV B22 proteins were synthesized at GenScript (Piscataway, NJ).

Techniques: Incubation, Transduction, Control, Inhibition, Clone Assay, Enzyme-linked Immunospot, Staining, Expressing

Journal: PLoS Pathogens

Article Title: T Cell Inactivation by Poxviral B22 Family Proteins Increases Viral Virulence

doi: 10.1371/journal.ppat.1004123

Figure Lengend Snippet: A) 4 female RM were inoculated i.b. with 2×10 5 PFU of MPXV US2003 (WT) or MPXVΔ197 on day 0. Whole blood, BAL, and PBMC samples were taken on indicated dpi. 2 RM infected with MPXVUS2003, WT-4 and WT-3, were euthanized at 12 and 24 dpi, respectively. The remaining WT-infected were euthanized on days 37 and 38 pi. Animals infected with MPXVΔ197 were euthanized at 41 and 42 dpi. B) Average nighttime body temperature (7PM to 7AM) as determined by biotelemetry transmitters for RM infected with WT (black) or MPXVΔ197 (red) (mean +/− SEM). P = 0.0007 (area under curve (AUC), F-test). C) and D) Viral loads determined by qPCR in BAL ( C ) and whole blood ( D ) of WT (blue) or MPXVΔ197 (red)-infected RM. P = 0.003 (AUC, F-test) and P<0.0001 (AUC, F-test) for BAL and whole blood, respectively. E) Number of skin lesions in WT (blue) or MPXVΔ197 (red)-infected RM. The p -value for the AUC comparison is P = 0.0003 (F-test). F) Poxvirus-specific antibody titers were determined by ELISA using VACV as antigen. The titers were not statistically different between WT and MPXVΔ197 cohorts.

Article Snippet: Codon-optimized sequences of the C-terminal 3×FLAG (DYKDHDGDYKDHDIDYKDDDDK) fusions of MPXV197 and VARV B22 proteins were synthesized at GenScript (Piscataway, NJ).

Techniques: Infection, Comparison, Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: T Cell Inactivation by Poxviral B22 Family Proteins Increases Viral Virulence

doi: 10.1371/journal.ppat.1004123

Figure Lengend Snippet: A) PBMC from WT (blue) and MPXVΔ197 (red)-infected RM were infected with VACV (MOI of 0.3) for 18 h. The background-subtracted frequency of poxvirus-responsive CD4+ and CD8+ T cells was determined by ICCS for TNFα and IFNγ. The differences were statistically significant at day 21 (P = 0.0063, F-test) for CD4+ T cells and at day 14 (P = 0.0069, F-test) for CD8+ T cells. B) The total frequency of CD4+ and CD8+ relative to day 0 as determined by flow cytometry is shown. The frequencies were not statistically different between WT and MPXVΔ197 cohorts. C) The percentage of CD4+ and CD8+ T cells relative to day 0 responding to anti-CD3 stimulation was determined by ICCS for IFNγ and TNFα. PBMC from WT (blue) or MPXVΔ197 (red) infected animals were stimulated with plate-bound αCD3 Ab for 6 h. The differences were statistically significant at day 14 (P = 0.0065, two-tailed t-test) for CD8+ T cells.

Article Snippet: Codon-optimized sequences of the C-terminal 3×FLAG (DYKDHDGDYKDHDIDYKDDDDK) fusions of MPXV197 and VARV B22 proteins were synthesized at GenScript (Piscataway, NJ).

Techniques: Infection, Flow Cytometry, Two Tailed Test