multiple lactobacillus strains l acidophilus atcc 4356  (ATCC)

Journal: bioRxiv

Article Title: Development of an antibody fused with an antimicrobial peptide targeting Pseudomonas aeruginosa: a new approach to prevent and treat bacterial infections

doi: 10.1101/2022.12.28.522163

Figure Lengend Snippet: (A) In vitro killing of P. aeruginosa PA14 mediated in the presence of PMN and Complement (OPKA) by VSX. The lot#-472 is representative of a Mab able to bind P. aeruginosa without any detectable OPKA. C=Complement. HI C=Heat Inactivated Complement. PMN=polymorphonuclear leukocytes. Abs=Antibodies. (B) Acute lung infection model . Challenge dose: 2×10 6 CFUs. Inoculation: intra nasal, 10 6 CFUs in each nostril. Mice=10/group (two experiments with 5 animal/group each time). Mab were injected intraperitoneally 4 hours post infection. Dose of the Mabs: VSX=15mg/kg. Control Mab (against Clostridioides difficile ): 15mg/kg. P. Value=0,04 measured by Log-Rank test.

Article Snippet: In particular, one member of this class, cathelicidin-BF , highlighted by one derivative - P297, demonstrated potent MIC values against multiple P. aeruginosa ATCC strains, low hemolytic activity, and a resistance to killing mammalian cells.

Techniques: In Vitro, Hi-C, Infection, Injection, Control

Journal: bioRxiv

Article Title: Development of an antibody fused with an antimicrobial peptide targeting Pseudomonas aeruginosa: a new approach to prevent and treat bacterial infections

doi: 10.1101/2022.12.28.522163

Figure Lengend Snippet: ( A ) Workflow for identification of an AMP to deploy in the construction of an ADC. ( B ) Antimicrobial peptide P297 showed rapid bactericidal activity in a time-kill assay using P. aeruginosa ATCC 27853. A growth control was compared to P297 at 0.5, 1, 2, 4, 8, or 16x MIC of peptide or to ciprofloxacin at 8x its MIC. Note that the 4x, 8x, 16x, and Ciprofloxacin 8x MICs all overlap. ( C ) Calcein leakage . Mechanism of action for P297 likely involves membrane disruption as assessed by measuring calcein leakage using DOPE/DOPG liposomes. Various concentrations of peptide, from 0.05– 100 µg/ml, were incubated with liposomes for a given amount of time (5-45 min). Release of calcein was assessed by measuring an increase in fluorescence at 530 nm compared to a non-peptide reference. Peptide concentrations above 1 µg/ml resulted in a measurable increase in fluorescence. ( D ) Assessment of resistance rates for P297 compared to colistin using two different procedures. ( E ) Comparison between wild type P. aeruginosa ATCC strain 27853 and two resistant mutants to P297 indicates differences in drug sensitivity as well as phenotypic differences. Mutant strains demonstrated decreased sensitivity to P297.

Article Snippet: In particular, one member of this class, cathelicidin-BF , highlighted by one derivative - P297, demonstrated potent MIC values against multiple P. aeruginosa ATCC strains, low hemolytic activity, and a resistance to killing mammalian cells.

Techniques: Activity Assay, Time-Kill Assay, Control, Membrane, Disruption, Liposomes, Incubation, Fluorescence, Comparison, Mutagenesis

Journal: bioRxiv

Article Title: Development of an antibody fused with an antimicrobial peptide targeting Pseudomonas aeruginosa: a new approach to prevent and treat bacterial infections

doi: 10.1101/2022.12.28.522163

Figure Lengend Snippet: ( A ) Synthesis of antibody-AMP conjugates using sortase ligation. VSX was expressed in Expi293 cells with a (GS) 15 flexible linker and a sortase acceptor tag (LPETGGSG) present at the C-terminus of both the light chain and the heavy chain. Then, a (GGG)-modified antimicrobial peptide AMP (AMP, for example P297) was covalently added via incubation with recombinantly produced sortase for a target DAR of 4. ( B ) Size exclusion chromatography (SEC-HPLC) of VSX (red line) and VSX conjugate (blue line) indicating an earlier shift in elution time for the modified construct compared to the starting antibody. The peak width and height is similar between the constructs indicating a relative homogeneity in sortase modification. ( C ) In vitro killing activity of VSX conjugates. P. aeruginosa ATCC strains 27853 or 39324 were treated with VSX conjugates with a DAR of ~4 containing AMP peptides 271, 293, 294, 295, or 297 and conjugate IgG concentrations that result in 50% killing are recorded. ( D ) VSX-1 (VSX with peptide P297 and a DAR of ~4) was characterized by several assays including in vitro bactericidal activity, opsonic activity, hemolytic activity and cytotoxic activity.

Article Snippet: In particular, one member of this class, cathelicidin-BF , highlighted by one derivative - P297, demonstrated potent MIC values against multiple P. aeruginosa ATCC strains, low hemolytic activity, and a resistance to killing mammalian cells.

Techniques: Ligation, Modification, Incubation, Produced, Size-exclusion Chromatography, Construct, In Vitro, Activity Assay

Journal: bioRxiv

Article Title: Development of an antibody fused with an antimicrobial peptide targeting Pseudomonas aeruginosa: a new approach to prevent and treat bacterial infections

doi: 10.1101/2022.12.28.522163

Figure Lengend Snippet: Mixed microbial killing assay. P. aeruginosa (blue), E. coli (red) and K. pneumoniae (green) were co-cultured overnight, diluted and then subjected to the specified agent for two hours. Killing was assessed visually. (left) Peptide P297 alone killing of P. aeruginosa and E. coli . (middle) In the absence of complement or immune cells, VSX alone had no killing effect. (right) VSX-1 demonstrated more rapid and complete killing of P. aeruginosa .

Article Snippet: In particular, one member of this class, cathelicidin-BF , highlighted by one derivative - P297, demonstrated potent MIC values against multiple P. aeruginosa ATCC strains, low hemolytic activity, and a resistance to killing mammalian cells.

Techniques: Cell Culture

Journal: bioRxiv

Article Title: Development of an antibody fused with an antimicrobial peptide targeting Pseudomonas aeruginosa: a new approach to prevent and treat bacterial infections

doi: 10.1101/2022.12.28.522163

Figure Lengend Snippet: MIC for both meropenem and colistin was determined alone and then varying concentrations of P297 were titrated to measure the effect on MIC towards P. aeruginosa ATCC 27853. ( A ) Examination of MIC for meropenem in the presence of different concentrations of P297. ( B ) Same as ( A ), except colistin was used as the antibiotic. Not all antibiotics demonstrated synergy; for example, the aminoglycoside tobramycin did not exhibit an enhanced MIC in the presence of P297.

Article Snippet: In particular, one member of this class, cathelicidin-BF , highlighted by one derivative - P297, demonstrated potent MIC values against multiple P. aeruginosa ATCC strains, low hemolytic activity, and a resistance to killing mammalian cells.

Techniques:

Journal: bioRxiv

Article Title: Development of an antibody fused with an antimicrobial peptide targeting Pseudomonas aeruginosa: a new approach to prevent and treat bacterial infections

doi: 10.1101/2022.12.28.522163

Figure Lengend Snippet: Neutralization of P. aeruginosa LPS activity in vitro. Binding of either VSX (blue) or actoxumab (grey) to LPS in a developed and optimized cell-based LPS neutralization assay. HEK-Blue LPS detection Kit was ordered from Invivogen to investigate the ability of VSX to neutralize endotoxin activity of extracted P. aeruginosa LPS on HEK-blue cells. In this case, endotoxin present in the media or standard is sensed by TLR4 leading to the activation of NF-kB and the production of SEAP in the supernatant. When supernatant is combined with QUANTI-Blue, this activation can be visualized and compared to a standard curve. 0.5 EU/ml P. aeruginosa LPS serotype was used for all assays.

Article Snippet: In particular, one member of this class, cathelicidin-BF , highlighted by one derivative - P297, demonstrated potent MIC values against multiple P. aeruginosa ATCC strains, low hemolytic activity, and a resistance to killing mammalian cells.

Techniques: Neutralization, Activity Assay, In Vitro, Binding Assay, Activation Assay

Journal: bioRxiv

Article Title: Development of an antibody fused with an antimicrobial peptide targeting Pseudomonas aeruginosa: a new approach to prevent and treat bacterial infections

doi: 10.1101/2022.12.28.522163

Figure Lengend Snippet: ( A ) Neutropenic animals co-administered VSX-1 and bacteria (ATCC 27853). CFU burden as measured in the lung at eight hours. Co-administration of 10 µg of VSX-1 resulted in a multi-log reduction in bacterial burden, with reduction to the limit of detection upon administering 200 µg of ADC. ( B ) Neutropenic animals were infected with P. aeruginosa (ATCC 27853) and were treated 1 hour post-infection with either vehicle or VSX-1 (200 µg). CFU burden was measured in the lungs just before treatment (pre-treatment) and in the lungs at eight hours post-injection. ( C ) Acute lung infection model with P. aeruginosa PA14 (2×10 6 CFU/animal, 10 6 in each nostril), C57/Bl6, 10 animals/group (two experiments with 5 animal/group each time), intranasal inoculation, intraperitoneal dosing (15 mg/kg) 4 hours post-infection.

Article Snippet: In particular, one member of this class, cathelicidin-BF , highlighted by one derivative - P297, demonstrated potent MIC values against multiple P. aeruginosa ATCC strains, low hemolytic activity, and a resistance to killing mammalian cells.

Techniques: Bacteria, Infection, Injection

Journal: bioRxiv

Article Title: Development of an antibody fused with an antimicrobial peptide targeting Pseudomonas aeruginosa: a new approach to prevent and treat bacterial infections

doi: 10.1101/2022.12.28.522163

Figure Lengend Snippet: ( A ) Activity of VSX-2 compared to VSX-1 as measured by the (bacterial) Killing Assay, hemolysis of red blood cells (mean lytic concentration (MLC)) and toxicity to mammalian cells (CC 50 ). The bactericidal activity of VSX-2 is similar, but slightly lower, than that of VSX-1, with a lower DAR, and with a similar inability to lyse RBCs or kill mammalian cells. ( B ) VSX-2 has similar activity in vivo in the acute lung infection model with P. aeruginosa PA14 (2 × 10 6 CFUs/animal, 10 6 CFUs in each nostril), C57/Bl6, 25 animals/group (five experiments with 5 animal/group each time), intranasal inoculation, intraperitoneal dosing (15 mg/kg) 4 hours post-infection.

Article Snippet: In particular, one member of this class, cathelicidin-BF , highlighted by one derivative - P297, demonstrated potent MIC values against multiple P. aeruginosa ATCC strains, low hemolytic activity, and a resistance to killing mammalian cells.

Techniques: Activity Assay, Concentration Assay, In Vivo, Infection