Validation of Ligand Tetramers for the Detection of Antigen-Specific Lymphocytes.

The study of Ag-specific lymphocytes has been a key advancement in immunology over the past few decades. The development of multimerized probes containing Ags, peptide:MHC complexes, or other ligands was one innovation allowing the direct study of Ag-specific lymphocytes by flow cytometry. Although these types of study are now common and performed by thousands of laboratories, quality control and assessment of probe quality are often minimal. In fact, many of these types of probe are made in-house, and protocols vary between laboratories. Although peptide:MHC multimers can often be obtained from commercial sources or core facilities, few such services exist for Ag multimers. To ensure high quality and consistency with ligand probes, we have developed an easy and robust multiplexed approach using commercially available beads able to bind Abs specific for the ligand of interest. Using this assay, we have sensitively assessed the performance of peptide:MHC and Ag tetramers and have found considerable batch-to-batch variability in performance and stability over time more easily than using murine or human cell-based assays. This bead-based assay can also reveal common production errors such as miscalculation of Ag concentration. This work could set the stage for the development of standardized assays for all commonly used ligand probes to limit laboratory-to-laboratory technical variation and experimental failure caused by probe underperformance.

Cross-protective antibodies against common endemic respiratory viruses.

Respiratory syncytial virus (RSV), human metapneumovirus (HMPV), and human parainfluenza virus types one (HPIV1) and three (HPIV3) can cause severe disease and death in immunocompromised patients, the elderly, and those with underlying lung disease. A protective monoclonal antibody exists for RSV, but clinical use is limited to high-risk infant populations. Hence, therapeutic options for these viruses in vulnerable patient populations are currently limited. Here, we present the discovery, in vitro characterization, and in vivo efficacy testing of two cross-neutralizing monoclonal antibodies, one targeting both HPIV3 and HPIV1 and the other targeting both RSV and HMPV. The 3 × 1 antibody is capable of targeting multiple parainfluenza viruses; the MxR antibody shares features with other previously reported monoclonal antibodies that are capable of neutralizing both RSV and HMPV. We obtained structures using cryo-electron microscopy of these antibodies in complex with their antigens at 3.62 Å resolution for 3 × 1 bound to HPIV3 and at 2.24 Å for MxR bound to RSV, providing a structural basis for in vitro binding and neutralization. Together, a cocktail of 3 × 1 and MxR could have clinical utility in providing broad protection against four of the respiratory viruses that cause significant morbidity and mortality in at-risk individuals.