Journal: Cell Reports Medicine

Article Title: A recombinant bovine adenoviral mucosal vaccine expressing mycobacterial antigen-85B generates robust protection against tuberculosis in mice

doi: 10.1016/j.xcrm.2021.100372

Figure Lengend Snippet: Diagrammatic representation of the gene cassette of the Ag85B-p25 epitope of Mycobacterium tuberculosis with or without the autophagy-inducing peptide C5 (AIP-C5) and the resultant BAdv vectors The 2 peptides were separated by the AlaAlaAla linker. The gene cassette was under the control of the cytomegalovirus (CMV) promoter and the bovine growth hormone (BGH) polyadenylation (PA) signal. The drawings are not to scale. 85, Ag85B-p25 epitope; C5, AIP-C5; LTR & RTR, the left and right terminal repeats; ΔE1, deletion of the early region 1; ΔE3, deletion of the early region 3. qPCR validation of Ag85B epitope and C5 epitope and the gene cassette for HAdv 85C5 vectors and vaccines are shown in A and S1B respectively.

Article Snippet: Gene constructs containing the immunogenic epitope Ag85B-p25 of Mtb without (85) or with AIP-C5 (85C5) were codon-optimized for rodent expression and synthesized commercially (GenScript Biotech Corporation, Piscataway, NJ).

Techniques: Control, Biomarker Discovery, Vaccines

Journal: Cell Reports Medicine

Article Title: A recombinant bovine adenoviral mucosal vaccine expressing mycobacterial antigen-85B generates robust protection against tuberculosis in mice

doi: 10.1016/j.xcrm.2021.100372

Figure Lengend Snippet: Replication-defective BAdv 85C5 vaccine induces a robust transcriptome in mouse DCs compared to HAdv 85C5 vaccine (A) Heatmaps of differentially expressed genes (DEGs) among DCs infected with BAdv- or HAdv-derived vaccines or vectors are shown (n = 2). Additional DEGs are shown in , and . (B–E) Gene expression analysis using ClusterProfiler (Novogene) indicated as KEGG (Kyoto Encyclopedia of Genes and Genomes) profiles followed by Gene Ontology (GO), cellular components (CC), and biological processes (BP). (F) Heatmaps of transcripts derived from RNA-seq expressed as FPKMs (fragments per kilobase per million mapped reads; n = 2); BAdv 85C5 vaccine upregulated genes involved in antigen processing are reproduced to the left and enriched genes are highlighted in blue (p < 0.0001, n = 2) (see also Figure S2 ). (G) Partial listing of DEGs are shown and those associated with antigen processing or endosome sorting are highlighted.

Article Snippet: Gene constructs containing the immunogenic epitope Ag85B-p25 of Mtb without (85) or with AIP-C5 (85C5) were codon-optimized for rodent expression and synthesized commercially (GenScript Biotech Corporation, Piscataway, NJ).

Techniques: Infection, Derivative Assay, Vaccines, Gene Expression, RNA Sequencing

Journal: Cell Reports Medicine

Article Title: A recombinant bovine adenoviral mucosal vaccine expressing mycobacterial antigen-85B generates robust protection against tuberculosis in mice

doi: 10.1016/j.xcrm.2021.100372

Figure Lengend Snippet: Replication-defective BAdv 85C5 vaccine is rapidly internalized by mouse DCs CD11c + DCs purified from bone marrow of WT-C57BL/6 mice were infected with the Cy3-labeled BAdv 85C5 vaccine or control vector (10 7 PFU/10 6 DCs), followed by antibody staining for intracellular localization using confocal microscopy at 4 h post-infection. Cy3-BAdv vector or Cy3-BAdv 85C5 vaccine-infected DCs were incubated for a 4-h infection, followed by fixation and staining using specific antibodies to (A) microtubule associate light chain 3 (LC3) autophagosome marker, (B) lysosome associated membrane protein-1 (LAMP1), (C) galectin3 ( Lgals-3 ), and (D) Lgals-8 followed anti-immunoglobulin G (IgG) Alexa Fluor 488 and DAPI nuclear stain. Panels show Cy3 (red), Alexa Fluor 488 (green), and merged images with or without DAPI nuclear stain. Colocalization (inset) analyzed using confocal microscopy and Nikon N90 microscope with MetaView software. Cytosolic (white arrow) and nuclear localization (yellow arrow) of Cy3-labeled vector or vaccines are indicated. Bar graphs to the right show percentage of virus-containing endosomes colocalizing with antibodies calculated per DC and averaged for 20 DCs in triplicates per experiments done twice. ∗p < 0.01 t test. Isotype stains are shown in Figure S5 .

Article Snippet: Gene constructs containing the immunogenic epitope Ag85B-p25 of Mtb without (85) or with AIP-C5 (85C5) were codon-optimized for rodent expression and synthesized commercially (GenScript Biotech Corporation, Piscataway, NJ).

Techniques: Purification, Infection, Labeling, Control, Plasmid Preparation, Staining, Confocal Microscopy, Incubation, Marker, Membrane, Microscopy, Software, Vaccines, Virus

Journal: Cell Reports Medicine

Article Title: A recombinant bovine adenoviral mucosal vaccine expressing mycobacterial antigen-85B generates robust protection against tuberculosis in mice

doi: 10.1016/j.xcrm.2021.100372

Figure Lengend Snippet: Replication-defective BAdv 85C5 vaccine enhances autophagy-dependent and -independent antigen presentation in mouse DCs and human macrophages (A) WT-C57BL/6- or ATG7 KO-C57BL/6-derived DCs were infected with vaccines or control vectors (10 7 plaque-forming units [PFUs]/10 6 DCs) followed by overlay using BB7 CD4 T cells specific for the Ag85B-p25 epitope in the context of MHC class II. IL-2 in supernatants at 18 h post-infection was determined using sandwich ELISA. (B) Antigen presentation in vaccine- or vector-infected mouse macrophages (10 7 PFU/10 6 macrophages) is shown. (C and D) Human peripheral blood mononuclear cell (PBMC)-derived CD14 + macrophages were infected with vaccines or control vectors followed by an overlay with F9A6 T cells specific for an Ag85B epitope in the context of human leukocyte antigen-DR1 (HLA-DR1) for antigen presentation. Macrophages were untreated or treated with 3-methyladenine (50 μM) before infection to inhibit autophagy. (E) Time-dependent antigen presentation by human macrophages infected with BAdv- or HAdv-derived vaccines. Horizontal line shows IL-2 levels of T cells incubated over naive macrophages. (F and G) CD14 + mouse bone marrow-derived macrophages were infected with 10 6 PFUs of vectors and vaccines indicated for 4 h, followed by co-infection with Mtb (Erdman strain; MOI = 1), washing, and incubation. Macrophage lysates were plated for CFU counts at indicated time points. For all of the panels, 1 of 2–3 separate experiments shown. ∗p < 0.01; ∗∗p < 0.009 1-way ANOVA with Tukey’s test.

Article Snippet: Gene constructs containing the immunogenic epitope Ag85B-p25 of Mtb without (85) or with AIP-C5 (85C5) were codon-optimized for rodent expression and synthesized commercially (GenScript Biotech Corporation, Piscataway, NJ).

Techniques: Immunopeptidomics, Derivative Assay, Infection, Vaccines, Control, Sandwich ELISA, Plasmid Preparation, Incubation

Journal: Cell Reports Medicine

Article Title: A recombinant bovine adenoviral mucosal vaccine expressing mycobacterial antigen-85B generates robust protection against tuberculosis in mice

doi: 10.1016/j.xcrm.2021.100372

Figure Lengend Snippet: Replication-defective BAdv 85C5 vaccine enhances galectin and cathepsin-dependent antigen presentation in mouse DCs and human macrophages C57BL/6 mouse-derived DCs were infected with vaccines or control vectors, washed, and processed. (A) At 18 h post-infection, lysates collected in Trizol were used for qPCR using primers for endosome trafficking genes enriched during transcriptomics ( Figure 2 ) (∗≥1.4-fold increase in mRNA expression, n = 2, 2 experiments). (B) At 18 h post-infection, lysates collected in anti-protease buffer analyzed using specific antibodies and Wes Simple blot protocol . Densitometry panels shown. (C and D) DCs and macrophages were subjected to siRNA versus Lgals-3 and Lgals-8 or scrambled controls and at 18 h, infected using vaccines or vectors for 4 h. Washed DCs were overlaid using BB7 CD4 T cells, and 18 h later, supernatants assayed for IL-2 using sandwich ELISA (triplicate wells of DCs per group and 2 independent experiments). (E) Lysates collected at 18 h post-infection were used for qPCR using primers for cathepsins enriched during transcriptomics ( Figure 2 ) (∗≥1.4-fold increase in mRNA expression, n = 2, 2 experiments). (F) Lysates collected at 18 h post-infection were analyzed using specific antibodies and Wes Simple blot protocol . (G) WT-DCs or ATG7 KO-DCs DCs were left untreated or treated with cathepsin inhibitors as indicated, followed by vaccine or vector infection and antigen presentation. NALLM, N -acetyl- l -leucyl- l -leucyl- l -methional; calpeptin; E64, each at 30 μM; triplicate wells of DCs per group and 2 independent experiments. ∗p < 0.01, ∗∗p < 0.006, 1-way ANOVA with Tukey’s post-test.

Article Snippet: Gene constructs containing the immunogenic epitope Ag85B-p25 of Mtb without (85) or with AIP-C5 (85C5) were codon-optimized for rodent expression and synthesized commercially (GenScript Biotech Corporation, Piscataway, NJ).

Techniques: Immunopeptidomics, Derivative Assay, Infection, Vaccines, Control, Expressing, Sandwich ELISA, Plasmid Preparation

Journal: Cell Reports Medicine

Article Title: A recombinant bovine adenoviral mucosal vaccine expressing mycobacterial antigen-85B generates robust protection against tuberculosis in mice

doi: 10.1016/j.xcrm.2021.100372

Figure Lengend Snippet: BAdv 85C5 mucosal vaccine protects mice against aerosolized Mtb either alone or as a booster following BCG vaccine (A) Female or male C57BL/6 mice, 4–6 weeks old, were nasally instilled using 1 dose each of vaccines or vectors at 10 7 PFUs. After 3 days, mice were sacrificed and bronchoalveolar lavage (BAL) cells and lung tissue suspended in Trizol and subjected to qPCR for gene expression (∗≥1.4-fold increase in mRNA expression, n = 2, 2 independent experiments). (B) Female or male C57BL/6 mice, 4–6 weeks old, were mock-inoculated or vaccinated subcutaneously with BCG. At 7 days post-immunization, animals received a single intranasal booster vaccination with 10 7 PFUs of BAdv, BAdv 85C5 , or BAdv vector control. Three weeks later, mice were aerosol challenged with Mtb ∼100 CFU of Erdman using a Glas-Col inhalation apparatus. Four weeks later, mice were sacrificed for Mtb counts, followed by analysis of the lungs and spleen for T cell profiles ( Figure 7 ). (C) BAdv 85C5 reduces the lungs and spleen burden of Mtb (∗∗p < 0.007, ∗∗∗p < 0.004; 2-way ANOVA with Dunnett’s post-test; n = 10, 5 mice per group each from 2 independent experiments plotted). (D) Flow cytometry data presented for 1 of 2 similar experiments integrated in (C). Lungs and spleen of mice collected post-Mtb challenge were stained using Ag85-p25 epitope-specific MHC class II tetramer (NIH tetramer core facility, Emory University) and analyzed using flow cytometry (n = 3 from the same group of mice, B and C; ∗∗∗∗p < 0.001; ordinary 1-way ANOVA). Arrowheads indicate BAdv 85C5 induced T cell expansion.

Article Snippet: Gene constructs containing the immunogenic epitope Ag85B-p25 of Mtb without (85) or with AIP-C5 (85C5) were codon-optimized for rodent expression and synthesized commercially (GenScript Biotech Corporation, Piscataway, NJ).

Techniques: Vaccines, Gene Expression, Expressing, Plasmid Preparation, Control, Aerosol, Flow Cytometry, Staining

Journal: Cell Reports Medicine

Article Title: A recombinant bovine adenoviral mucosal vaccine expressing mycobacterial antigen-85B generates robust protection against tuberculosis in mice

doi: 10.1016/j.xcrm.2021.100372

Figure Lengend Snippet: BAdv 85C5 booster induces stronger cytokine-positive T cells and effector (T EM ) and memory T cells (T CM ) in BCG-vaccinated mice following Mtb challenge (A–D) Lungs and spleens of mice vaccinated and challenged as in Figure 6 B were stained for CD4 and CD8 T cells expressing IFN-γ and IL-2 and analyzed using flow cytometry (∗∗∗∗p < 0.001; ordinary 1-way ANOVA). Arrowheads indicate BAdv 85C5 -induced T cell expansion. Flow cytometry data presented for 1 of 2 similar experiments integrated in Figure 6 C. (E–H) The lungs and spleen of mice vaccinated and challenged as in Figure 6 B were stained for CD4 and CD8 T EM and T CM followed by flow cytometric analysis (∗p < 0.01, ∗∗∗,∗∗∗∗p < 0.001, ordinary 1-way ANOVA). Arrows indicate BAdv 85C5 -induced T cell expansion. T RMs (CD4 + CD103 + CD69 + ) on day 21 for post-vaccination (vax) and day 60 post-Mtb challenge are shown in Figure S7 .

Article Snippet: Gene constructs containing the immunogenic epitope Ag85B-p25 of Mtb without (85) or with AIP-C5 (85C5) were codon-optimized for rodent expression and synthesized commercially (GenScript Biotech Corporation, Piscataway, NJ).

Techniques: Staining, Expressing, Flow Cytometry

Journal: Infection and Immunity

Article Title: Vaccine-Specific Immune Responses against Mycobacterium ulcerans Infection in a Low-Dose Murine Challenge Model

doi: 10.1128/IAI.00753-19

Figure Lengend Snippet: Summary of putative M. ulcerans vaccines tested in a murine model of BU infection a

Article Snippet: TABLE 1 Vaccine type Description of components M. ulcerans challenge dose (strain) b Challenge model Efficacy compared to BCG Reference(s) DNA based pCDNA3 vector encoding Hsp65 10 4 AFB (1615 ATCC 35840) Tail Less protective 52 DNA based V1Jns.tPA vector encoding Ag85A 3 × 10 4 AFB (5150) or 10 5 AFB (04-855) Footpad Less protective 32 , 33 DNA based Primary vaccination with V1Jns.tPA plasmid encoding mycolactone polyketide domains and boosted with recombinant domain proteins emulsified in Gerbu adjuvant 10 5 AFB (1615) Footpad Less protective 49 Viral Vesicular stomatitis virus replicon particles expressing M. ulcerans codon optimized antigens MUL_2232 and MUL_3720 30 μl of 2.8 × 10 5 CFU/ml stock (8.4 × 10 3 CFU/dose) (S1013) Footpad Less protective 108 Subunit MUL2232 and MUL3720 adjuvanted with GLA-SE (EM408) 1.5 × 10 6 or 1.5 × 10 5 CFU (S1013) Footpad Less protective 34 Live cell M. ulcerans 10 6.3 or 10 4.3 viable bacteria Footpad Less protective 109 Live cell M. marinum 10 5 bacteria (1615) Footpad More protective c 35 Live cell recombinant M. marinum expressing Ag85A (on vector) 10 5 bacteria (1615) Footpad More protective c 35 Live cell recombinant M. bovis BCG expressing Ag85A (on vector pMV261) 10 5 bacteria (1615) Footpad More protective 36 Live cell recombinant M. bovis BCG expressing Ag85B-EsxH fusion protein Ag85A (on vector pMV261) 10 5 bacteria (1615) Footpad More protective 37 Inactivated whole cell Mycolactone-negative M. ulcerans (strain 5114) 4 log 10 or 3 log 10 CFU (98-912) Footpad Less protective 25 Inactivated whole cell Mycolactone-deficient attenuated M. ulcerans (strain ATCC 19423) 10 6 bacteria (TMC1615) Footpad Not compared 54 Inactivated whole cell Formalin-treated M. ulcerans (strain TMC1615) 10 6 bacteria (TMC1615) Footpad Not compared 54 Inactivated whole cell Dewaxed M. ulcerans (strain TMC1615) 10 6 bacteria (TMC1615) Footpad Not compared 54 Phage Mycobacteriophage D29 (therapeutic vaccine) 5.5 log 10 AFB (1615) Footpad Not comparable 53 Open in a separate window a No test of whether the bacterial culture used for challenge was assessed for mycolactone production before infection was mentioned with any of the vaccines. b AFB, acid-fast bacilli. c Vaccine was more protective than the BCG vaccine; however, all mice eventually developed footpad swelling.

Techniques: Vaccines, Infection, Plasmid Preparation, Recombinant, Adjuvant, Virus, Expressing, Bacteria

Journal: Infection and Immunity

Article Title: Vaccine-Specific Immune Responses against Mycobacterium ulcerans Infection in a Low-Dose Murine Challenge Model

doi: 10.1128/IAI.00753-19

Figure Lengend Snippet: Summary of putative M. ulcerans vaccines tested in a murine model of BU infection a

Article Snippet: While improving the immunogenicity of BCG may be a promising route, there are also some drawbacks; exposure to environmental mycobacteria is believed to decrease the efficacy of the BCG vaccine, and administration in areas where people have been exposed to BCG may be problematic ( 38 ). table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Vaccine type Description of components M. ulcerans challenge dose (strain) b Challenge model Efficacy compared to BCG Reference(s) DNA based pCDNA3 vector encoding Hsp65 10 4 AFB (1615 ATCC 35840) Tail Less protective 52 DNA based V1Jns.tPA vector encoding Ag85A 3 × 10 4 AFB (5150) or 10 5 AFB (04-855) Footpad Less protective 32 , 33 DNA based Primary vaccination with V1Jns.tPA plasmid encoding mycolactone polyketide domains and boosted with recombinant domain proteins emulsified in Gerbu adjuvant 10 5 AFB (1615) Footpad Less protective 49 Viral Vesicular stomatitis virus replicon particles expressing M. ulcerans codon optimized antigens MUL_2232 and MUL_3720 30 μl of 2.8 × 10 5 CFU/ml stock (8.4 × 10 3 CFU/dose) (S1013) Footpad Less protective 108 Subunit MUL2232 and MUL3720 adjuvanted with GLA-SE (EM408) 1.5 × 10 6 or 1.5 × 10 5 CFU (S1013) Footpad Less protective 34 Live cell M. ulcerans 10 6.3 or 10 4.3 viable bacteria Footpad Less protective 109 Live cell M. marinum 10 5 bacteria (1615) Footpad More protective c 35 Live cell recombinant M. marinum expressing Ag85A (on vector) 10 5 bacteria (1615) Footpad More protective c 35 Live cell recombinant M. bovis BCG expressing Ag85A (on vector pMV261) 10 5 bacteria (1615) Footpad More protective 36 Live cell recombinant M. bovis BCG expressing Ag85B-EsxH fusion protein Ag85A (on vector pMV261) 10 5 bacteria (1615) Footpad More protective 37 Inactivated whole cell Mycolactone-negative M. ulcerans (strain 5114) 4 log 10 or 3 log 10 CFU (98-912) Footpad Less protective 25 Inactivated whole cell Mycolactone-deficient attenuated M. ulcerans (strain ATCC 19423) 10 6 bacteria (TMC1615) Footpad Not compared 54 Inactivated whole cell Formalin-treated M. ulcerans (strain TMC1615) 10 6 bacteria (TMC1615) Footpad Not compared 54 Inactivated whole cell Dewaxed M. ulcerans (strain TMC1615) 10 6 bacteria (TMC1615) Footpad Not compared 54 Phage Mycobacteriophage D29 (therapeutic vaccine) 5.5 log 10 AFB (1615) Footpad Not comparable 53 Open in a separate window a No test of whether the bacterial culture used for challenge was assessed for mycolactone production before infection was mentioned with any of the vaccines. b AFB, acid-fast bacilli. c Vaccine was more protective than the BCG vaccine; however, all mice eventually developed footpad swelling.

Techniques: Infection, Plasmid Preparation, Recombinant, Expressing

Journal: Infection and Immunity

Article Title: Vaccine-Specific Immune Responses against Mycobacterium ulcerans Infection in a Low-Dose Murine Challenge Model

doi: 10.1128/IAI.00753-19

Figure Lengend Snippet: Summary of putative M. ulcerans vaccines tested in a murine model of BU infection a

Article Snippet: While improving the immunogenicity of BCG may be a promising route, there are also some drawbacks; exposure to environmental mycobacteria is believed to decrease the efficacy of the BCG vaccine, and administration in areas where people have been exposed to BCG may be problematic ( 38 ). table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Vaccine type Description of components M. ulcerans challenge dose (strain) b Challenge model Efficacy compared to BCG Reference(s) DNA based pCDNA3 vector encoding Hsp65 10 4 AFB (1615 ATCC 35840) Tail Less protective 52 DNA based V1Jns.tPA vector encoding Ag85A 3 × 10 4 AFB (5150) or 10 5 AFB (04-855) Footpad Less protective 32 , 33 DNA based Primary vaccination with V1Jns.tPA plasmid encoding mycolactone polyketide domains and boosted with recombinant domain proteins emulsified in Gerbu adjuvant 10 5 AFB (1615) Footpad Less protective 49 Viral Vesicular stomatitis virus replicon particles expressing M. ulcerans codon optimized antigens MUL_2232 and MUL_3720 30 μl of 2.8 × 10 5 CFU/ml stock (8.4 × 10 3 CFU/dose) (S1013) Footpad Less protective 108 Subunit MUL2232 and MUL3720 adjuvanted with GLA-SE (EM408) 1.5 × 10 6 or 1.5 × 10 5 CFU (S1013) Footpad Less protective 34 Live cell M. ulcerans 10 6.3 or 10 4.3 viable bacteria Footpad Less protective 109 Live cell M. marinum 10 5 bacteria (1615) Footpad More protective c 35 Live cell recombinant M. marinum expressing Ag85A (on vector) 10 5 bacteria (1615) Footpad More protective c 35 Live cell recombinant M. bovis BCG expressing Ag85A (on vector pMV261) 10 5 bacteria (1615) Footpad More protective 36 Live cell recombinant M. bovis BCG expressing Ag85B-EsxH fusion protein Ag85A (on vector pMV261) 10 5 bacteria (1615) Footpad More protective 37 Inactivated whole cell Mycolactone-negative M. ulcerans (strain 5114) 4 log 10 or 3 log 10 CFU (98-912) Footpad Less protective 25 Inactivated whole cell Mycolactone-deficient attenuated M. ulcerans (strain ATCC 19423) 10 6 bacteria (TMC1615) Footpad Not compared 54 Inactivated whole cell Formalin-treated M. ulcerans (strain TMC1615) 10 6 bacteria (TMC1615) Footpad Not compared 54 Inactivated whole cell Dewaxed M. ulcerans (strain TMC1615) 10 6 bacteria (TMC1615) Footpad Not compared 54 Phage Mycobacteriophage D29 (therapeutic vaccine) 5.5 log 10 AFB (1615) Footpad Not comparable 53 Open in a separate window a No test of whether the bacterial culture used for challenge was assessed for mycolactone production before infection was mentioned with any of the vaccines. b AFB, acid-fast bacilli. c Vaccine was more protective than the BCG vaccine; however, all mice eventually developed footpad swelling.

Techniques: Infection, Plasmid Preparation, Recombinant, Expressing