Optimized Expression of Full-Length IgG1 Antibody in a Common E. coli Strain

Multi-polypeptide proteins such as antibodies are difficult to express in prokaryotic systems such as E. coli due to the complexity of protein folding plus secretion. Thus far, proprietary strains or fermenter cultures have been required for appreciable yields. Previous studies have shown that expression of heterologous proteins in E. coli can be enhanced by the reduction of protein translation rates. In this paper, we demonstrate that useful quantities of full-length IgG can be expressed and purified from the common E. coli laboratory strain HB2151 in standard shaking culture using a simple strategy of reduced inducer concentration combined with delayed induction times to modulate translation rates. Purified IgG had only marginally reduced avidity compared to mammalian derived IgG. This indicates that this technique can be used to derive antibodies of potentially equal utility as those expressed in mammalian cell culture, particularly for applications where effector functions mediated by the glycosylated residues in the Fragment Crystallizable (Fc) portion of the immunoglobulin are not required

The diagnostic targeting of a carbohydrate virulence factor from M.Tuberculosis

10.1038/srep10281Scientific Reports

Lipid anti-lipid antibody responses correlate with disease activity in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder characterized by broad clinical manifestations including cardiovascular and renal complications with periodic disease flares and significant morbidity and mortality. One of the main contributing factors to the pathology of SLE is the accumulation and impaired clearance of immune complexes of which the principle components are host auto-antigens and antibodies. The contribution of host lipids to the formation of these autoimmune complexes remains poorly defined. The aim of the present study was to identify and analyze candidate lipid autoantigens and their corresponding anti-lipid antibody responses in a well-defined SLE patient cohort using a combination of immunological and biophysical techniques. Disease monitoring in the SLE cohort was undertaken with serial British Isles Lupus Assessment Group (BILAG) scoring. Correlations between specific lipid/anti-lipid responses were investigated as disease activity developed from active flares to quiescent during a follow up period. We report a significant negative correlation between anti-lipid antibodies for 24S-hydroxycholesterol, cardiolipin and phosphatidylserine with SLE disease activity. Taken together, these data suggest that lipid autoantigens represent a new family of biomarkers that can be employed to monitor disease activity plus the efficacy of therapeutic intervention in SLE

Generation and Characterization of a Novel Recombinant Antibody Against 15-Ketocholestane Isolated by Phage-Display

The employment of monoclonal antibodies (Mabs) to identify disease-associated biomarkers in clinical samples represents the underlying principle for many diagnostic tests. To date, these have been principally developed for protein targets with few reported applications for lipids due to their hydrophobicity and poor immunogenicity. Oxysterols represent a family of lipids implicated in diverse human diseases where Mab-based detection assays could have a profound effect on their utility as clinical biomarkers. These are usually identified in patients’ samples by mass- spectrometry based approaches. Here, we describe an antibody phage-library based screening methodology for generating a recombinant monoclonal antibody (RAb) targeting the oxysterol-15-ketocholestane (15-KA), a lipid implicated in multiple sclerosis and Autoimmune Encephalomyelitis (EAE). The antibody is highly specific for 15-KA and shows little or no binding activity for other closely related oxysterols. We employ RAb2E9 to address the controversy over whether 15-KA is a true biomarker for MS/EAE and show that 15-KA is undetectable in serum taken from mice with EAE using antibody based detection methodologies; a finding confirmed by mass-spectrometry analysis. This study demonstrates the technical feasibility of using phage display to isolate highly specific antibodies against poorly immunogenic, small molecule lipids

Leucocyte subset-specific type 1 interferon signatures in SLE and other immune-mediated diseases.

OBJECTIVES: Type 1 interferons (IFN-1) are implicated in the pathogenesis of systemic lupus erythematosus (SLE), but most studies have only reported the effect of IFN-1 on mixed cell populations. We aimed to define modules of IFN-1-associated genes in purified leucocyte populations and use these as a basis for a detailed comparative analysis. METHODS: CD4+ and CD8+ T cells, monocytes and neutrophils were purified from patients with SLE, other immune-mediated diseases and healthy volunteers and gene expression then determined by microarray. Modules of IFN-1-associated genes were defined using weighted gene coexpression network analysis. The composition and expression of these modules was analysed. RESULTS: 1150 of 1288 IFN-1-associated genes were specific to myeloid subsets, compared with 11 genes unique to T cells. IFN-1 genes were more highly expressed in myeloid subsets compared with T cells. A subset of neutrophil samples from healthy volunteers (HV) and conditions not classically associated with IFN-1 signatures displayed increased IFN-1 gene expression, whereas upregulation of IFN-1-associated genes in T cells was restricted to SLE. CONCLUSIONS: Given the broad upregulation of IFN-1 genes in neutrophils including in some HV, investigators reporting IFN-1 signatures on the basis of whole blood samples should be cautious about interpreting this as evidence of bona fide IFN-1-mediated pathology. Instead, specific upregulation of IFN-1-associated genes in T cells may be a useful biomarker and a further mechanism by which elevated IFN-1 contributes to autoimmunity in SLE.SMF holds a Translational Medicine and Therapeutics PhD studentship from the Wellcome Trust and GlaxoSmithKline and has also received funding for this work from the Addenbrooke’s Charitable Trust. KGCS is the Khoo Oon Teik Professor of Nephrology, National University of Singapore. Singapore recruitment was supported by the Khoo Investigator Grant from the Duke-NUS Graduate Medical School, Singapore, and by National Medical Research Council of Singapore grants (NMRC/1164/2008 and IRG07nov089). This work was also supported by UK National Institute of Health Research Cambridge Biomedical Research Centre, the Lupus Research Institute (Distinguished Innovator Award, KGCS), the Medical Research Council UK (programme grant MR/L019027/1) and the Wellcome Trust (programme grant 083650/Z/07/Z and project grant 094227/Z/10/Z). The Cambridge Institute for Medical Research is in receipt of Wellcome Trust Strategic Award 079895.This is the final version of the article. It first appeared from BMJ Group via https://doi.org/10.1136/rmdopen-2015-00018

Atypical B cells and impaired SARS-CoV-2 neutralization following heterologous vaccination in the elderly

Suboptimal responses to a primary vaccination course have been reported in the elderly, but there is little information regarding the impact of age on responses to booster third doses. Here, we show that individuals 70 years or older (median age 73, range 70-75) who received a primary two-dose schedule with AZD1222 and booster third dose with mRNA vaccine achieve significantly lower neutralizing antibody responses against SARS-CoV-2 spike pseudotyped virus compared with those younger than 70 (median age 66, range 54-69) at 1 month post booster. Impaired neutralization potency and breadth post third dose in the elderly is associated with circulating "atypical" spike-specific B cells expressing CD11c and FCRL5. However, when considering individuals who received three doses of mRNA vaccine, we did not observe differences in neutralization or enrichment in atypical B cells. This work highlights the finding that AdV and mRNA COVID-19 vaccine formats differentially instruct the memory B cell response