Here, we describe the discovery of a naturally occurring human antibody (Ab), FluA-20, that recognizes a new site of vulnerability on the hemagglutinin (HA) head domain and reacts with most influenza A viruses. Structural characterization of FluA-20 with H1 and H3 head domains revealed a novel epitope in the HA trimer interface, suggesting previously unrecognized dynamic features of the trimeric HA protein. The critical HA residues recognized by FluA-20 remain conserved across most subtypes of influenza A viruses, which explains the Ab's extraordinary breadth. The Ab rapidly disrupted the integrity of HA protein trimers, inhibited cell-to-cell spread of virus in culture, and protected mice against challenge with viruses of H1N1, H3N2, H5N1, or H7N9 subtypes when used as prophylaxis or therapy. The FluA-20 Ab has uncovered an exceedingly conserved protective determinant in the influenza HA head domain trimer interface that is an unexpected new target for anti-influenza therapeutics and vaccines.

Class I HLA molecules mark infected cells for immune targeting by presenting pathogen-encoded peptides on the cell surface. Characterization of viral peptides unique to infected cells is important for understanding CD8(+) T cell responses and for the development of T cell-based immunotherapies. Having previously reported a series of West Nile virus (WNV) epitopes that are naturally presented by HLA-A*02:01, in this study we generated TCR mimic (TCRm) mAbs to three of these peptide/HLA complexes-the immunodominant SVG9 (E protein), the subdominant SLF9 (NS4B protein), and the immunorecessive YTM9 (NS3 protein)-and used these TCRm mAbs to stain WNV-infected cell lines and primary APCs. TCRm staining of WNV-infected cells demonstrated that the immunorecessive YTM9 appeared several hours earlier and at 5- to 10-fold greater density than the more immunogenic SLF9 and SVG9 ligands, respectively. Moreover, staining following inhibition of the TAP demonstrated that all three viral ligands were presented in a TAP-dependent manner despite originating from different cellular compartments. To our knowledge, this study represents the first use of TCRm mAbs to define the kinetics and magnitude of HLA presentation for a series of epitopes encoded by one virus, and the results depict a pattern whereby individual epitopes differ considerably in abundance and availability. The observations that immunodominant ligands can be found at lower levels and at later time points after infection suggest that a reevaluation of the factors that combine to shape T cell reactivity may be warranted.

BACKGROUND Identification of species-specific trypanosome molecules is important for laboratory- and field-based research into epidemiology and disease diagnosis. Although Trypanosoma congolense is the most important trypanosome pathogen of cattle in Africa, no species-specific molecules found in infective bloodstream forms (BSF) of the parasites have been identified, thus limiting development of diagnostic tests. METHODS Immuno-mass spectrometric methods were used to identify a protein that is recognized by a T. congolense-specific monoclonal antibody (mAb) Tc6/42.6.4. The identified molecule was expressed as a recombinant protein in E. coli and was tested in several immunoassays for its ability to interact with the mAb. The three dimensional structure of the protein was modeled and compared to crystal- and NMR-structures of the homologous proteins from T. cruzi and T. brucei respectively, in order to examine structural differences leading to the different immunoreactivity of the T. congolense molecule. Enzyme-linked immunosorbent assays (ELISA) were used to measure antibodies produced by trypanosome-infected African cattle in order to assess the potential for use of T. congolense calflagin in a serodiagnostic assay. RESULTS The antigen recognized by the T. congolense-specific mAb Tc6/42.6.4 was identified as a flagellar calcium-binding protein, calflagin. The recombinant molecule showed immunoreactivity with the T. congolense-specific mAb confirming that it is the cognate antigen. Immunofluorescence experiments revealed that Ca2+ modulated the localization of the calflagin molecule in trypanosomes. Structural modelling and comparison with calflagin homologues from other trypanosomatids revealed four non-conserved regions on the surface of the T. congolense molecule that due to differences in surface chemistry and structural topography may form species-specific epitopes. ELISAs using the recombinant calflagin as antigen to detect antibodies in trypanosome-infected cattle showed that the majority of cattle had antibody responses. Area under the Receiver-Operating Characteristic (ROC) curves, associated with host IgG and IgM, were calculated to be 0.623 and 0.709 respectively, indicating a positive correlation between trypanosome infection and the presence of anti-calflagin antibodies. CONCLUSIONS While calflagin is conserved among different species of African trypanosomes, our results show that T. congolense calflagin possesses unique epitopes that differentiate this protein from homologues in other trypanosome species. MAb Tc6/42.6.4 has clear utility as a laboratory tool for identifying T. congolense. T. congolense calflagin has potential as a serodiagnostic antigen and should be explored further for its utility in antigen-detection assays for diagnosis of cattle infections.