The evolutionary distance of lampreys from humans in conjunction with their distinct antibody architecture is profound. Thus, lampreys may provide antibodies with specificity for antigens unrecognized by conventional mammalian antibodies. This study investigates lamprey based monoclonal variable lymphocyte receptor antibodies (VLRs) for purifying and identifying an antigen by tandem mass spectrometry. VLRs specific for clinically relevant cell populations were isolated. Subsequently, utilizing intrinsic VLR affinity, with or without covalent cross-linking molecules, for immunoprecipitating VLR protein antigens was tested. In one case, CD5 glycoprotein from Jurkat T cells was purified by a VLR; the antigen was identified by tandem mass spectrometry. Antibody specificity was validated by western blotting and flow cytometry. Furthermore, VLR binding to CD5 required multimerization of the antibody, indicating the individual VLR units likely bind antigen with low affinity. The study provides ‘proof of concept’ for human biomarker identification using novel lamprey monoclonal antibodies.

The evolutionary distance of lampreys from humans in conjunction with their distinct antibody architecture is profound. Thus, lampreys may provide antibodies with specificity for antigens unrecognized by conventional mammalian antibodies. This study investigates lamprey based monoclonal variable lymphocyte receptor antibodies (VLRs) for purifying and identifying an antigen by tandem mass spectrometry. VLRs specific for clinically relevant cell populations were isolated. Subsequently, utilizing intrinsic VLR affinity, with or without covalent cross-linking molecules, for immunoprecipitating VLR protein antigens was tested. In one case, CD5 glycoprotein from Jurkat T cells was purified by a VLR; the antigen was identified by tandem mass spectrometry. Antibody specificity was validated by western blotting and flow cytometry. Furthermore, VLR binding to CD5 required multimerization of the antibody, indicating the individual VLR units likely bind antigen with low affinity. The study provides ‘proof of concept’ for human biomarker identification using novel lamprey monoclonal antibodies.

Staphylococcus aureus remains an important pathogen, which, due to its capability to develop antimicrobial resistance, imposes an increasing threat to human health. Developing preventive means to decrease disease burden is a major aim. However, the development of an S. aureus vaccine, which would be one strategy to achieve such goals, has been complicated through limited understanding of the bacterium's pathogenic mechanisms. This work uses four approaches to address these limitations: Firstly, a reproducible RNA sequencing based method for the determination of gene transcription by S. aureus in vivo during mammalian infection. Secondly, examination of the impact of the bacterial transcription regulator 'Rsp' on the bacterium, which shows that mutations in this gene have profound functional and transcriptional impacts. Thirdly, by examining the in vivo transcription of multiple S. aureus strains during infection, proposing a 'core in vivo transcriptome' of induced genes under the conditions tested. Some of these genes are known to be involved in pathogenesis, others are not completely characterised and may represent suitable vaccine antigens. Finally, this work addresses limited understanding of S. aureus pathogenesis through defining transcriptional changes in vivo, which are induced by an altered immune response in immunised hosts. Together, this body of work contributes to the understanding of S. aureus pathogenesis and provides candidate antigens for future vaccine development.