B CELL EPITOPE MAPPING OF RIVAX, A CANDIDATE RICIN VACCINE ANTIGEN

Ricin toxin’s enzymatic A subunit (RTA) is a 267 amino acid RNA N-glycosidase that depurinates a conserved adenine residue of 28S rRNA, resulting in ribosome arrest and apoptosis. One of the leading subunit vaccine candidates for ricin is RiVax, a two point mutant (V76M, Y80A) of RTA. RiVax has proven to be safe in humans, however, it could not elicit a robust toxin-neutralizing antibody response. In order to redesign a potent ricin subunit vaccine candidate based on RTA, an immunological rationale has to be implemented. Protection against ricin is antibody mediated and hence generating a comprehensive B cell epitope map of ricin toxin would not only help in evaluating future ricin vaccine candidates in humans but also provides an immunological rationale for designing new vaccine candidates. Previous studies have shown that toxin neutralizing antibodies recognized four immunodominant regions on RTA i.e. four epitope clusters (namely cluster I to IV). RTA’s active site is surrounded by cluster III. Surprisingly, mAb IB2, which defines cluster III is the only antibody that recognized this immunodominant region. We previously showed that IB2 binds to helices C and G on the surface of RiVax. In this study, we sought to gain a better understanding of cluster III using a collection (21) of single domain antibodies (VHHs) that are derived from ricin immunized alpacas. To this end, I first produced and characterized RiVax (a safer version which is structurally identical to RTA) for its structural integrity since our main emphasis lies in identifying discontinuous/ conformational epitopes. Based on hydrogen exchange mass spectrometry (HX-MS) studies, VHHS recognized overlapping epitopes with four spatially distinct contact regions i.e. they were grouped into four subclusters (namely 3.1 to 3.4) within cluster III region. Subcluster 3.1 includes helices C and G. Subcluster 3.2 encompasses additionally helix B along with helices C and G. While subcluster 3.3 consists of helices B and G, subcluster 3.4 includes helices C, E and strand h. Of the 21 antibodies that we analyzed, only two, namely V1D3 and V6D4 have shown toxin neutralizing activity (TNA). Both neutralizing antibodies have strong binding affinity (sub nanomolar range) to the toxin and engaged a common secondary structural element, namely helix G as part of their epitope. The second part of my dissertation focusses on fibroblast growth factor-1 (FGF-1), a member of β-trefoil family of proteins. FGFs regulate a number of developmental process including mitogenesis, angiogenesis and homeostasis etc. Due to their wide range of biological activities, FGFs has been of interest in several clinical applications. In particular, wound healing has generated considerable interest. Studies have shown that several polyanions (sulfated and phosphorylated) have increased the thermal stability of FGF-1 by 15-30oC. In this study, we sought to identify the binding sites of the polyanions. In particular, we studied two sulfated (heparin, low MW heparin) and two phosphorylated (phytic acid and ATP) polyanions. Using HX-MS, we examined the local dynamics as well as binding sites of the polyanions. For local dynamics, we identified strand 4 and 5 and the turn connecting them to be most flexible which agrees with previous NMR studies. On the other hand, strands 8, 9 and 10 appear to be more rigid which is consistent with crystallographic B factors as well as local dynamic studies conducted by NMR. Crystal studies have shown that heparin binds to N-terminal Asn18 and to C-terminal Lys105, Tryp107, Lys112, Lys113, Arg119, Pro121, Arg122, Gln127 and Lys128 indicating electrostatic forces as the dominant interactions. Heparin binding as determined by HX-MS is consistent with the crystallography data. We find other polyanions tested bind in a similar manner to heparin, primarily targeting the turns in the lysine rich C-terminal region of FGF-1 along with two distinct N-terminal regions that contains lysines and arginines/ histidines. This confirms the interactions between FGF-1 and polyanions are primary directed by electrostatics

High-Resolution Epitope Positioning of a Large Collection of Neutralizing and Nonneutralizing Single-Domain Antibodies on the Enzymatic and Binding Subunits of Ricin Toxin

We previously produced a heavy-chain-only antibody (Ab) VH domain (VHH)-displayed phage library from two alpacas that had been immunized with ricin toxoid and nontoxic mixtures of the enzymatic ricin toxin A subunit (RTA) and binding ricin toxin B subunit (RTB) (D. J. Vance, J. M. Tremblay, N. J. Mantis, and C. B. Shoemaker, J Biol Chem 288:36538–36547, 2013, https://doi.org/10.1074/jbc.M113.519207). Initial and subsequent screens of that library by direct enzyme-linked immunosorbent assay (ELISA) yielded more than two dozen unique RTA- and RTB-specific VHHs, including 10 whose structures were subsequently solved in complex with RTA. To generate a more complete antigenic map of ricin toxin and to define the epitopes associated with toxin-neutralizing activity, we subjected the VHH-displayed phage library to additional “pannings” on both receptor-bound ricin and antibody-captured ricin. We now report the full-length DNA sequences, binding affinities, and neutralizing activities of 68 unique VHHs: 31 against RTA, 33 against RTB, and 4 against ricin holotoxin. Epitope positioning was achieved through cross-competition ELISAs performed with a panel of monoclonal antibodies (MAbs) and verified, in some instances, with hydrogen-deuterium exchange mass spectrometry. The 68 VHHs grouped into more than 20 different competition bins. The RTA-specific VHHs with strong toxin-neutralizing activities were confined to bins that overlapped two previously identified neutralizing hot spots, termed clusters I and II. The four RTB-specific VHHs with potent toxin-neutralizing activity grouped within three adjacent bins situated at the RTA-RTB interface near cluster II. These results provide important insights into epitope interrelationships on the surface of ricin and delineate regions of vulnerability that can be exploited for the purpose of vaccine and therapeutic development

A Collection of Single-Domain Antibodies that Crowd Ricin Toxin’s Active Site

This work is licensed under a Creative Commons Attribution 4.0 International License.In this report, we used hydrogen exchange-mass spectrometry (HX-MS) to identify the epitopes recognized by 21 single-domain camelid antibodies (VHHs) directed against the ribosome-inactivating subunit (RTA) of ricin toxin, a biothreat agent of concern to military and public health authorities. The VHHs, which derive from 11 different B-cell lineages, were binned together based on competition ELISAs with IB2, a monoclonal antibody that defines a toxin-neutralizing hotspot (“cluster 3”) located in close proximity to RTA’s active site. HX-MS analysis revealed that the 21 VHHs recognized four distinct epitope subclusters (3.1–3.4). Sixteen of the 21 VHHs grouped within subcluster 3.1 and engage RTA α-helices C and G. Three VHHs grouped within subcluster 3.2, encompassing α-helices C and G, plus α-helix B. The single VHH in subcluster 3.3 engaged RTA α-helices B and G, while the epitope of the sole VHH defining subcluster 3.4 encompassed α-helices C and E, and β-strand h. Modeling these epitopes on the surface of RTA predicts that the 20 VHHs within subclusters 3.1–3.3 physically occlude RTA’s active site cleft, while the single antibody in subcluster 3.4 associates on the active site’s upper rim.National Institutes of Allergy and Infectious Diseases, National Institutes of Health (HHSN272201400021C

High-Definition Mapping of Four Spatially Distinct Neutralizing Epitope Clusters on RiVax, a Candidate Ricin Toxin Subunit Vaccine

RiVax is a promising recombinant ricin toxin A subunit (RTA) vaccine antigen that has been shown to be safe and immunogenic in humans and effective at protecting rhesus macaques against lethal-dose aerosolized toxin exposure. We previously used a panel of RTA-specific monoclonal antibodies (MAbs) to demonstrate, by competition enzyme-linked immunosorbent assay (ELISA), that RiVax elicits similar serum antibody profiles in humans and macaques. However, the MAb binding sites on RiVax have yet to be defined. In this study, we employed hydrogen exchange-mass spectrometry (HX-MS) to localize the epitopes on RiVax recognized by nine toxin-neutralizing MAbs and one nonneutralizing MAb. Based on strong protection from hydrogen exchange, the nine MAbs grouped into four spatially distinct epitope clusters (namely, clusters I to IV). Cluster I MAbs protected RiVax's α-helix B (residues 94 to 107), a protruding immunodominant secondary structure element known to be a target of potent toxin-neutralizing antibodies. Cluster II consisted of two subclusters located on the “back side” (relative to the active site pocket) of RiVax. One subcluster involved α-helix A (residues 14 to 24) and α-helices F-G (residues 184 to 207); the other encompassed β-strand d (residues 62 to 69) and parts of α-helices D-E (154 to 164) and the intervening loop. Cluster III involved α-helices C and G on the front side of RiVax, while cluster IV formed a sash from the front to back of RiVax, spanning strands b, c, and d (residues 35 to 59). Having a high-resolution B cell epitope map of RiVax will enable the development and optimization of competitive serum profiling assays to examine vaccine-induced antibody responses across species

A Collection of Single-Domain Antibodies that Crowd Ricin Toxin’s Active Site

In this report, we used hydrogen exchange-mass spectrometry (HX-MS) to identify the epitopes recognized by 21 single-domain camelid antibodies (VHHs) directed against the ribosome-inactivating subunit (RTA) of ricin toxin, a biothreat agent of concern to military and public health authorities. The VHHs, which derive from 11 different B-cell lineages, were binned together based on competition ELISAs with IB2, a monoclonal antibody that defines a toxin-neutralizing hotspot (“cluster 3”) located in close proximity to RTA’s active site. HX-MS analysis revealed that the 21 VHHs recognized four distinct epitope subclusters (3.1–3.4). Sixteen of the 21 VHHs grouped within subcluster 3.1 and engage RTA α-helices C and G. Three VHHs grouped within subcluster 3.2, encompassing α-helices C and G, plus α-helix B. The single VHH in subcluster 3.3 engaged RTA α-helices B and G, while the epitope of the sole VHH defining subcluster 3.4 encompassed α-helices C and E, and β-strand h. Modeling these epitopes on the surface of RTA predicts that the 20 VHHs within subclusters 3.1–3.3 physically occlude RTA’s active site cleft, while the single antibody in subcluster 3.4 associates on the active site’s upper rim