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Intact Transition Epitope Mapping – Serological Inspection by Epitope EXtraction (ITEM – SIX)

ABSTRACT: Precision medicine requests accurate serological inspections to precisely stratify patients for targeted treatment. Intact transition epitope mapping analysis proved seroconversion of a model organism's serum when spiked with a monoclonal murine anti-ovalbumin antibody with epitope-resolution. Isolation of the IgG fraction from blood serum applied two consecutive protein precipitation steps followed by ultrafiltration and resulted in an ESI-MS analysis-ready IgG preparation. For epitope mapping by epitope extraction, the ovalbumin antigen was digested with trypsin. After desalting, the peptide mixture was added to the ESI-MS-ready IgG preparation from converted serum and the solution was incubated to form an immune complex between the ovalbumin-derived epitope peptide and the anti-ovalbumin antibody. Then, the entire mixture of proteins and peptides was directly electrosprayed. Sorting of ions in the mass spectrometer's gas phase, dissociation of the immune complex ions by collision induced dissociation, and recording of the epitope peptide ion which had been released from the immune complex revealed the presence of the anti-ovalbumin antibody in converted serum. Mass determination of the complex-released epitope peptide ion with isotope resolution is highly accurate, guaranteeing high specificity of this novel seroconversion analysis approach which is termed Intact Transition Epitope Mapping – Serological Inspections by Epitope EXtraction (ITEM – SIX).

INSTRUMENT(S): Waters instrument model

ORGANISM(S): Gallus Gallus (chicken)

SUBMITTER: Michael Kreutzer

LAB HEAD: Prof. Dr. Michael O. Glocker

PROVIDER: PXD040754 | Pride | 2023-03-31

REPOSITORIES: Pride

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Project description:Recently, several neutralizing anti-HIV antibodies have been isolated from memory B cells of HIV-infected individuals. However, despite extensive evidence of B-cell dysfunction in HIV disease, little is known about the cells from which these rare HIV-specific antibodies originate. Accordingly, HIV envelope gp140 and CD4 or co-receptor (CoR) binding site (bs) mutant probes were used to evaluate HIV-specific responses in the peripheral blood B cells of individuals at various stages of infection. In contrast to non-HIV responses, HIV-specific responses against gp140 were enriched within abnormal B cells, namely activated and exhausted memory subsets, which are largely absent in the blood of uninfected individuals. Responses against the CoRbs (a poorly-neutralizing epitope) arose early whereas those against the CD4bs (a well-characterized neutralizing epitope) were delayed and infrequent. Enrichment of the HIV-specific response within resting memory B cells, the predominant subset in uninfected individuals, did occur in certain infected individuals who maintained low levels of plasma viremia and immune activation with or without antiretroviral therapy. These findings were corroborated by transcriptional profiles. Taken together, our findings provide valuable insight into virus-specific B-cell responses in HIV infection and demonstrate that memory B-cell abnormalities may contribute to the ineffectiveness of the antibody response in infected individuals. HIV-specific responses against gp140 were enriched within abnormal B cells, namely activated (AM) and exhausted (tissue-like; TLM) memory subsets, which are largely absent in the blood of uninfected individuals. These responses are highest during the early stage of HIV infection, significantly decreased following the initiation of antiretroviral therapy (ART), and most importantly, enriched in normal resting memory B cells (RM) when HIV viremia and immune activation are controlled either naturally or as a result of ART. These HIV-specific B cells (AM and TLM) and resting memory B cells (RM) were sorted from peripheral blood mononuclear cells (PBMCs) of 6 HIV infected individuals. In addition, gp140-specific IgG+ B cells were sorted from individuals with either a strong (n= 6) or weak (n= 6) pro-resting memory profile. TaqMan gene expression assay was performed on these HIV-specific B cells and B cell subset. The array consisted of 29 genes.

Project description:Resiquimod is a nucleoside analog belonging to the imidazoquinoline family of compounds which is known to signal through Toll-like receptor 7. Resiquimod treatment has been demonstrated to inhibit the development of allergen induced asthma in experimental models. Despite this demonstrated effectiveness, little is known about the molecular events responsible for this effect. The aim of the present study was to elucidate the molecular processes which were altered following resiquimod treatment and antigen challenge in a mouse model of allergic asthma. Employing microarray analysis, we have characterized the 'asthmatic' transcriptome of the murine lung and determined that it includes genes involved in: the control of cell cycle progression, airway remodelling, the complement and coagulation cascades, and chemokine signalling. We have demonstrated that systemic resiquimod administration resulted in the recruitment of NK cells to the lungs of the mice, although no causal relationship between NK cell recruitment and treatment efficacy was found. Furthermore, results of our studies demonstrated that resiquimod treatment resulted in the normalization of the expression of genes involved with airway remodelling and chemokine signalling, and in the modulation of the expression of genes including cytokines and chemokines, adhesion molecules, and B-cell related genes, involved in several aspects of immune function and antigen presentation. Overall, our findings identified several genes, important in the development of asthma pathology, that were normalized following resiquimod treatment thus improving our understanding of the molecular consequences of resiquimod treatment in the lung milieu. Experiment Overall Design: A total of 18 samples, from 6 sets of biological replicates, were analyzed. 9 A/J and 9 C57BL/6 mice were divided into three equal groups. All animals were sensitized to ovalbumin, one group received PBS aerosol challenges, the other two received 1% ovalbumin aerosol challenges. Of the two ovalbumin challenged groups, one recieved resiquimod 24hours before each challenge.

			Action	DRS
	F417101.dat	Other
	F417101.mgf	Mgf
	checksum.txt	Txt
	solution_0a.raw.zip	Raw
	solution_0b.raw.zip	Raw

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Intact Transition Epitope Mapping – Serological Inspection by Epitope EXtraction (ITEM – SIX)

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