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IP-MALDI-MS assay LOD for VP40 by targeting two peptide variants

ABSTRACT: We developed a quantitative assay for carefully selected VP40 peptide variants. We selected two peptides, LGPGIPDHPLR from EBOV VP40 and LRPILLPGK from BDBV VP40 to generate pan-peptide variants antibodies. Potential structural difference in virion particles affect the efficiency of VP40 liberation during sample inactivation, and the sequence variations also affect the affinity of peptide against the pan-variants antibody. Therefore, we quantified the mass contents of VP40 in EBOV and SUDV VLPs and used the VLPs from each species to build the species-specific standard curves. This improves the accuracy of VP40 quantitation. The feasibility of using miniature mass spectrometer to collect the tandem MS spectra for both peptides was validated.

INSTRUMENT(S): autoflex

ORGANISM(S): Ebolavirus

SUBMITTER: Qingbo Shu

LAB HEAD: Tony Hu

PROVIDER: PXD026048 | Pride | 2021-11-02

REPOSITORIES: Pride

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Project description:Human papilloma (HPV) virus-like particle (VLP) vaccines were recently licensed. Though neutralizing antibody titers are thought to be the main effectors of protection against infection, early predictors of long-term efficacy are not yet defined and a comprehensive understanding of innate and adaptive immune responses to vaccination is still lacking. Here, microarrays were used to compare the gene expression signature in HPV-16 L1 VLP-stimulated PBMC from 17 vaccine and 4 placebo recipients before vaccination, and 1 month after receiving the second immunization. Vaccination with HPV-16 L1 VLP was associated with modulation of genes involved in the inflammatory/defense response, cytokine, interferon and cell cycle pathways in VLP-stimulated PBMC. In addition, there was up-regulation of probesets associated with cytotoxic (GZMB, TNFSF10) and regulatory (INDO, CTLA4) activities. The strongest correlations with neutralizing antibody titers were found for cyclin d2 (CCND2) and galectin (LGALS2). Twenty-two differentially expressed probesets were selected for confirmation by RT-PCR in an independent sample set. Agreement with the microarray data was seen for over two-thirds of these probesets. Up-regulation of immune/defense response genes by VLP, in particular interferon-induced genes was observed in PBMC collected prior to vaccination, with many of these genes being further induced following vaccination. In conclusion, we used gene expression profiling to identify important innate and adaptive response related- genes induced by vaccination with HPV VLP. Further studies are needed to identify gene expression signatures of immunogenicity and long-term protection with potential utility in prediction of long-term HPV vaccination outcomes in clinical trials. Experiment Overall Design: Microarrays (Affymetrix Human Focus) were used to compare the gene expression signature in PBMCs stimulated for 3 days with media alone, Sf9/baculovirus insect cell lysate, and HPV-16 L1 VLP expressed from baculovirus-infected Sf9 insect cells from 17 vaccine and 4 placebo recipients before vaccination, and 1 month after receiving the second immunization (2 months after the initial immunization. Post-vaccination baculovirus sample for Pt078 and pre-vaccination baculovirus sample for Pt082 failed QC.

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IP-MALDI-MS assay LOD for VP40 by targeting two peptide variants

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