ABSTRACT
The quantitation of antibody responses is a critical requirement for the successful development of vaccines and therapeutics that often relies on the use of standardized reference materials to determine relative quantities within biological samples. The validity of comparing responses across assays using arbitrarily defined reference values is therefore limited. We developed a generalizable method known as MASCALE (Mass Spectrometry Enabled Conversion to Absolute Levels of ELISA Antibodies) for absolute quantitation of antibodies by calibrating ELISA reference sera using mass spectrometry. Levels of proteotypic peptides served as a proxy for human IgG, allowing the conversion of responses from arbitrary values to absolute amounts. Applications include comparison of binding assays at two separate laboratories and evaluation of cross-clade magnitude-breadth responses induced by an investigational HIV-1 vaccine regimen. MASCALE addresses current challenges in the interpretation of immune responses in clinical trials and expands current options available to make suitable comparisons across different settings.
ABSTRACT
Epitope characterization is critical for elucidating the mechanism of action of drug candidates. However, traditional high-resolution epitope mapping techniques are not well suited for screening numerous drug candidates recognizing a similar target. Here, we use Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) to explore the conformational impact of diverse drug molecules binding on Hemagglutinin (HA), the major surface antigen of influenza viruses. We optimized a semi-automated HDX-MS workflow to systematically probe distantly related HA subtypes in complex with 4 different drug candidates, ranging from a monoclonal antibody to a small synthetic peptide. This fast, cost-effective HDX-MS epitope mapping approach accurately determined the main antigenic site in all cases. Moreover, our studies reveal distinct changes in the local conformational dynamics of HA associated to the molecular mechanism of neutralization, establishing a marker for broad anti-HA activity. Taken together, these findings highlight the potential for HDX-MS epitope mapping-based screening to identify promising candidates against HA at early stages of drug discovery.
Subject(s)
Epitope Mapping/methods , Hemagglutinins/metabolism , Hydrogen Deuterium Exchange-Mass Spectrometry/methods , Influenza, Human/drug therapy , Drug Discovery/methods , Hemagglutinins/immunology , Humans , Pharmaceutical Preparations/metabolism , Protein BindingABSTRACT
The identification of human broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem revitalized hopes of developing a universal influenza vaccine. Using a rational design and library approach, we engineered stable HA stem antigens ("mini-HAs") based on an H1 subtype sequence. Our most advanced candidate exhibits structural and bnAb binding properties comparable to those of full-length HA, completely protects mice in lethal heterologous and heterosubtypic challenge models, and reduces fever after sublethal challenge in cynomolgus monkeys. Antibodies elicited by this mini-HA in mice and nonhuman primates bound a wide range of HAs, competed with human bnAbs for HA stem binding, neutralized H5N1 viruses, and mediated antibody-dependent effector activity. These results represent a proof of concept for the design of HA stem mimics that elicit bnAbs against influenza A group 1 viruses.
