Journal: Infection and Immunity

Article Title: Construction and characterization of novel Mycobacterium tuberculosis -derived triple and quadruple knockout vaccines against tuberculosis

doi: 10.1128/iai.00500-25

Figure Lengend Snippet: Rational deletion of genes reduces the intracellular survival and increases the processing of live vaccines through PL fusion and autophagy. ( A ) Deletion of genes in Mtb reduces intracellular viability/growth. BMDMs from naïve C57BL/6 mice were activated with IFN-γ and infected (MOI 1:1) with Mtb strains H37Rv, DKO, TKO-Z, TKO-D, or QKO and incubated. After 1, 4, and 8 days post-infection, cells were washed, lysed, and plated for viable colony counts (CFUs). Data represent the mean ± SD CFUs from triplicate. ( B ) Deletion of genes in Mtb leads to efficient processing through PL fusion. BMDMs from naïve C57BL/6 mice were infected with (a–e) rfpH37Rv, rfpDKO, rfpTKO-D, rfpTKO-Z, and rfpQKO (MOI = 1:1) for 4 h, washed, incubated for 24 h, and stained with primary antibodies to phagosomal maturation marker Rab7, followed by staining with FITC (green) conjugated secondary antibody. Red fluorescent Mtb colocalizing with Rab7 antibodies was scored using a Nikon TiE Fluorescence Microscope and Metaview Deconvolution Software. ( C ) Graph showing percent colocalization of Mtb with Rab7. Percent colocalization was determined by counting 50 macrophages per well, each with 1–3 mycobacteria, and averaging counts from triplicate chambers. One of three similar experiments is shown. ( D ) Deletion of genes in Mtb leads to efficient processing through autophagy. BMDMs from naïve C57BL/6 mice were infected with (a–e) rfpH37Rv, rfpDKO, rfpTKO-D, rfpTKO-Z, and rfpQKO (MOI = 1:1) for 4 h, washed, incubated for 24 h, and stained with primary antibodies to autophagy marker LC3, followed by staining with FITC (green) conjugated secondary antibody. Red fluorescent Mtb colocalizing with LC3 antibodies was scored using a Nikon TiE Fluorescence Microscope and Metaview Deconvolution Software. ( E ) The graph shows the percent colocalization of Mtb with LC3. Percent colocalization was determined by counting 50 macrophages per well, each with 1–3 mycobacteria, and averaging counts from triplicate chambers. One of three similar experiments is shown. Statistical significance was calculated using Student’s t-test. P values below 0.05 ( P < 0.05) are considered significant.

Article Snippet: Mouse IFN-γ ELISpot Plus plates (Catalog #3321-4APT-10, MABTECH Inc., Cincinnati, OH) were washed three times with sterile PBS and then blocked with culture media (RMPI with 10% FBS).

Techniques: Vaccines, Infection, Incubation, Staining, Marker, Fluorescence, Microscopy, Software

Journal: Infection and Immunity

Article Title: Construction and characterization of novel Mycobacterium tuberculosis -derived triple and quadruple knockout vaccines against tuberculosis

doi: 10.1128/iai.00500-25

Figure Lengend Snippet: Rational deletion of genes in Mtb increases the immunogenicity of vaccine strains. ( A ) In vitro antigen presentation. BMDMs from naïve C57BL/6 mice were infected with BCG and Mtb strains (H37Rv, DKO, TKO-D, TKO-Z, and QKO) (MOI = 1:5) for 4 h and cocultured with BB7 T-cell hybridoma specific for Ag85B 241-256 peptide. After 16 h, culture fluids were collected and assayed for IL-2 levels released by the BB7 cells in response to Ag85B peptide. ( B ) Ex vivo immunogenicity to vaccine strains. C57BL/6 mice were vaccinated with BCG, DKO, TKO-D, TKO-Z, and QKO vaccine strains (1 × 10 6 subcutaneously) and the control H37Rv. After 30 days of post-immunization, mice were euthanized, and spleens were isolated. Splenocytes (2.5 × 10 5 /well) were plated and stimulated in vitro for 48 h with Mtb H37Rv whole-cell lysate (20 µg/mL). Supernatants from cultures were collected, and IFN-γ, (a) IL-1β, (b) IL-2, (c) IL-12 (d), and TNF (e) levels were determined by ELISA. ELISpot analysis for IFN-γ-producing splenocytes in vaccinated mice (f). C57BL/6 mice were vaccinated with BCG, DKO, TKO-D, TKO-Z, and QKO vaccine strains and control H37Rv (1 × 10 6 subcutaneously). After 30 days post-immunization, mice were euthanized, and spleens were isolated. Splenocytes (2.5 × 10 5 /well) were plated and stimulated with a combination of Ag85B and CFP-10 peptides in vitro for 48 h. Ag85B/CFP-10 responsive IFN-γ-producing spleen cells were spotted using IFN-γ ELISpot plates following the manufacturer’s protocols. Statistical significance was calculated using Student’s t-test. P values below 0.05 ( P < 0.05) were considered significant.

Article Snippet: Mouse IFN-γ ELISpot Plus plates (Catalog #3321-4APT-10, MABTECH Inc., Cincinnati, OH) were washed three times with sterile PBS and then blocked with culture media (RMPI with 10% FBS).

Techniques: Immunopeptidomics, In Vitro, Infection, Ex Vivo, Control, Isolation, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: iScience

Article Title: Extensive evolution and T cell escape by SARS-CoV-2 in a 2.5-year persistent infection of an immunocompromised host

doi: 10.1016/j.isci.2026.114917

Figure Lengend Snippet: Magnitude and specificity of T cell memory responses generated by 899-day infection (A) Ex vivo IFNγ-ELISpot well images. DMSO control unstimulated wells and wells stimulated with overlapping peptides covering S1 and S2 region of Spike (duplicates shown), or single peptides S51 and S134. (B) Total magnitude of SARS-CoV-2-specific memory T cell response to structural proteins Spike, membrane (M), nucleoprotein (NP) and open reading frame 3a (ORF3a) and RTC proteins (NSP7, NSP12 polymerase, and NSP13 helicase) colored by protein targeted and measured after persistent infection (>900 days). (C and D) Total magnitude of SARS-CoV-2-specific T cell response (C) or T cell response to Structural and RTC proteins (D) in pre-pandemic samples (pre-August 2019), in exposed healthcare workers (HCW) who remained seronegative, including abortive infections, and in HCW with laboratory confirmed SARS-CoV-2 infections (samples 4 months post-exposure/infection in June to July 2020) for comparison to T cell response in persistently infected patient. (E) Ratio of the magnitude of the T cell response to RTC/structural T cells. Percentage of cohort with a response above 1 (stronger response to RTC than structural proteins) shown below. (F) Magnitude of T cell response to a pool of epitopes from Flu, EBV, and CMV. (B–E) Subset of data previously published in Swadling et al. (A–F) IFNγ-ELISpot. (C and D) Box and Whisker, Tukey. (C–F) Statistical analysis was performed using Kruskal-Wallis tests with Dunn’s correction. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001; ∗∗∗∗ p < 0.0001. (E and F) Bars, geomean.

Article Snippet: Plates were washed in double-distilled H 2 O and left to dry overnight before being read on the AID classic ELISpot plate reader (Immunospot S6 Universal M2 ELISpot Reader).

Techniques: Generated, Infection, Ex Vivo, Enzyme-linked Immunospot, Control, Membrane, Comparison, Whisker Assay

Journal: iScience

Article Title: Extensive evolution and T cell escape by SARS-CoV-2 in a 2.5-year persistent infection of an immunocompromised host

doi: 10.1016/j.isci.2026.114917

Figure Lengend Snippet: Failure of host T cells to recognize emerging virus (A) Ex vivo magnitude of the T cell response to individual ancestral sequence peptides in which mutation arose over persistent infection by IFNγ-ELISpot. (B) Magnitude of the CD4 and CD8 T cell responses after 10-day in vitro peptide expansion with individual ancestral sequence peptides. Percentage of CD4 and CD8 producing IFNγ, TNF, or both are shown. (C) Magnitude of IFNγ+, IFNγ+TNF+, and CTV lo IFNγ+ CD4 and CD8 T cells after 8-day expansion with a pool of 12 peptides corresponding to ancestral sequence epitopes or variant sequence epitopes containing mutations in the virus isolated at day 899 of infection. (D) Magnitude of the IFNγ+TNF+ CD4 or CD8 T cell response after 10-day in vitro peptide stimulation with individual epitopes using ancestral sequence or variant sequence peptides for culture and re-stimulation on day 9. Summary data showing all T cell responses that were detectable (left) or shown for individual peptides (right) for CD4 then CD8 T cells. Duplicate and triplicate stimulations are shown for CD8 T cells for M14 and NSP2, respectively. (D) Bars, mean. Statistical analysis was performed using Kruskal-Wallis tests with Dunn’s correction. ∗ p ≤ 0.05; ∗∗∗ p ≤ 0.001. Peptide sequences shown in .

Article Snippet: Plates were washed in double-distilled H 2 O and left to dry overnight before being read on the AID classic ELISpot plate reader (Immunospot S6 Universal M2 ELISpot Reader).

Techniques: Virus, Ex Vivo, Sequencing, Mutagenesis, Infection, Enzyme-linked Immunospot, In Vitro, Variant Assay, Isolation