Advanced control strategies for the continuous production of monoclonal antibodies

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Extending Chemical Perturbations Of The Ubiquitin Fitness Landscape In A Classroom Setting [preprint]

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary time scales. Building on our previous work (Mavor et al. 2016), we used deep mutational scanning to determine how twelve new chemicals reveal novel mutational sensitivities of ubiquitin residues. We found sensitization of Lys63 in eight new conditions. In total, our experiments have uncovered a highly sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the Ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales

Extending chemical perturbations of the ubiquitin fitness landscape in a classroom setting reveals new constraints on sequence tolerance

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales

Dual gene activation and knockout screen reveals directional dependencies in genetic networks.

Understanding the direction of information flow is essential for characterizing how genetic networks affect phenotypes. However, methods to find genetic interactions largely fail to reveal directional dependencies. We combine two orthogonal Cas9 proteins from Streptococcus pyogenes and Staphylococcus aureus to carry out a dual screen in which one gene is activated while a second gene is deleted in the same cell. We analyze the quantitative effects of activation and knockout to calculate genetic interaction and directionality scores for each gene pair. Based on the results from over 100,000 perturbed gene pairs, we reconstruct a directional dependency network for human K562 leukemia cells and demonstrate how our approach allows the determination of directionality in activating genetic interactions. Our interaction network connects previously uncharacterized genes to well-studied pathways and identifies targets relevant for therapeutic intervention

IL-17 Production from T Helper 17, Mucosal-Associated Invariant T, and γδ Cells in Tuberculosis Infection and Disease.

IL-17-producing cells have been shown to be important in the early stages of Mycobacterium tuberculosis (Mtb) infection in animal models. However, there are very little data on the role of IL-17 in human studies of tuberculosis (TB). We recruited TB patients and their highly exposed contacts who were further categorized based on results from an IFN-γ-release assay (IGRA): (1) IGRA positive (IGRA+) at recruitment (latently TB infected), (2) IGRA negative (IGRA-) at recruitment and 6 months [non-converters (NC)], and (3) IGRA- at recruitment and IGRA+ at 6 months (converters). Whole blood was stimulated with mycobacterial antigens and analyzed using T helper (Th) 17 multiplex cytokine assays. Th17, Vγ9Vδ2+, and CD161++Vα7.2+ mucosal-associated invariant T (MAIT) cells were analyzed by flow cytometry. The majority of IL-17 was produced by CD26+CD4+ Th17 cells (median 71%) followed by γδ T cells (6.4%) and MAIT cells (5.8%). TB patients had a significantly lower proportion of Th17 cells and CD4+CD161+Vα7.2+ cells producing both IL-17 and IFN-γ compared to LTBI subjects. IGRA NC had significantly lower levels of CD26-CD4+ and CD8+ MAIT cells producing IL-17 compared to IGRA C but had significantly higher levels of IL-17A, IL-17F, IL-21, and IL-23 in ESAT-6/CFP-10-stimulated supernatants compared to IGRA C. These data provide new insights into the role of IL-17 and IL-17-producing cells at three key stages of the Mtb infection spectrum

Mycobacterium avium-intracellulare cellulitis occurring with septic arthritis after joint injection: a case report

BACKGROUND: Cellulitis caused by Mycobacterium avium-intracellulare has rarely been described. Mycobacterium avium-intracellulare is a rare cause of septic arthritis after intra-articular injection, though the causative role of injection is difficult to ascertain in such cases. CASE PRESENTATION: A 57-year-old with rheumatoid arthritis treated with prednisone and azathioprine developed bilateral painful degenerative shoulder arthritis. After corticosteroid injections into both acromioclavicular joints, he developed bilateral cellulitis centered over the injection sites. Skin biopsy showed non-caseating granulomas, and culture grew Mycobacterium avium-intracellulare. Joint aspiration also revealed Mycobacterium avium-intracellulare infection. CONCLUSION: Although rare, skin and joint infections caused by Mycobacterium avium-intracellulare should be considered in any immunocompromised host, particularly after intra-articular injection. Stains for acid-fast bacilli may be negative in pathologic samples even in the presence of infection; cultures of tissue specimens should always be obtained

Safety and immunogenicity of a novel 10-valent pneumococcal conjugate vaccine candidate in adults, toddlers, and infants in The Gambia-Results of a phase 1/2 randomized, double-blinded, controlled trial.

BACKGROUND: A more affordable pneumococcal conjugate vaccine (PCV) that provides comparable protection to current PCVs is needed to ensure sustainable access in resource-limited settings. Serum Institute of India Pvt. Ltd.'s PCV candidate (SIIPL-PCV) has the potential to meet this need as manufacturing efficiency has been optimized and the vaccine targets the most prevalent disease-causing serotypes in Africa and Asia. We report SIIPL-PCV's safety, tolerability, and immunogenicity in adults, toddlers, and infants in The Gambia. METHODS: This phase 1/2, randomized, double-blind trial sequentially enrolled 34 PCV-naive adults (18-40?years old), 112 PCV (Prevenar 13® [PCV13])-primed toddlers (12-15?months old), and 200 PCV-naive infants (6-8?weeks old), who were randomized (1:1) to receive SIIPL-PCV or a licensed comparator vaccine. Infants received three-doses of SIIPL-PCV or PCV13 at 6, 10, and 14?weeks of age co-administered with routine Expanded Program on Immunization (EPI) vaccines. Reactogenicity was solicited through seven-days post-vaccination; unsolicited adverse events (AEs) were assessed throughout the study. The safety and immunogenicity of a matching booster at 10-14?months of age were evaluated in a subset of 96 infants. Immune responses were evaluated post-primary and pre- and post-booster vaccinations. RESULTS: Reactogenicity was primarily mild-to-moderate in severity. In infants, the most common solicited reactions were injection-site tenderness and fever, with no meaningful treatment-group differences. There were no serious or severe vaccine-related AEs and no meaningful trends in SAEs, vaccine-related AEs, or overall AEs. Infant post-primary seroresponse rates (IgG level???0.35?µg/mL) were ?89% for all serotypes except 6A (79%) in the SIIPL-PCV group. IgG GMCs were >1?µg/mL for all serotypes in both SIIPL-PCV and PCV13 groups. Post-booster GMCs were comparable between groups. CONCLUSION: SIIPL-PCV was well-tolerated, had an acceptable safety profile, and was immunogenic for all vaccine serotypes. Results support the evaluation of SIIPL-PCV in a phase 3 non-inferiority trial. Clinicaltrials.gov: NCT02308540

Safety and immunogenicity of inactivated poliovirus vaccine when given with measles–rubella combined vaccine and yellow fever vaccine and when given via diff erent administration routes: a phase 4, randomised, non-inferiority trial in The Gambia

Background The introduction of the inactivated poliovirus vaccine (IPV) represents a crucial step in the polio eradication endgame. This trial examined the safety and immunogenicity of IPV given alongside the measles–rubella and yellow fever vaccines at 9 months and when given as a full or fractional dose using needle and syringe or disposable-syringe jet injector. Methods We did a phase 4, randomised, non-inferiority trial at three periurban government clinics in west Gambia. Infants aged 9–10 months who had already received oral poliovirus vaccine were randomly assigned to receive the IPV, measles–rubella, and yellow fever vaccines, singularly or in combination. Separately, IPV was given as a full intramuscular or fractional intradermal dose by needle and syringe or disposable-syringe jet injector at a second visit. The primary outcomes were seroprevalence rates for poliovirus 4–6 weeks post-vaccination and the rate of seroconversion between baseline and post-vaccination serum samples for measles, rubella, and yellow fever; and the post-vaccination antibody titres generated against each component of the vaccines. We did a per-protocol analysis with a non-inferiority margin of 10% for poliovirus seroprevalence and measles, rubella, and yellow fever seroconversion, and (⅓) log2 for log2-transformed antibody titres. This trial is registered with ClinicalTrials.gov, number NCT01847872. Findings Between July 10, 2013, and May 8, 2014, we assessed 1662 infants for eligibility, of whom 1504 were enrolled into one of seven groups for vaccine interference and one of four groups for fractional dosing and alternative route of administration. The rubella and yellow fever antibody titres were reduced by co-administration but the seroconversion rates achieved non-inferiority in both cases (rubella, –4·5% [95% CI –9·5 to –0·1]; yellow fever, 1·2% [–2·9 to 5·5]). Measles and poliovirus responses were unaff ected (measles, 6·8% [95% CI –1·4 to 14·9]; poliovirus serotype 1, 1·6% [–6·7 to 4·7]; serotype 2, 0·0% [–2·1 to 2·1]; serotype 3, 0·0% [–3·8 to 3·9]). Poliovirus seroprevalence was universally high (>97%) after vaccination, but the antibody titres generated by fractional intradermal doses of IPV did not achieve non-inferiority compared with full dose. The number of infants who seroconverted or had a four-fold rise in titres was also lower by the intradermal route. There were no safety concerns. Interpretation The data support the future co-administration of IPV, measles–rubella, and yellow fever vaccines within the Expanded Programme on Immunization schedule at 9 months. The administration of single fractional intradermal doses of IPV by needle and syringe or disposable-syringe jet injector compromises the immunity generated, although it results in a high post-vaccination poliovirus seroprevalence