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We had previously shown that three anti–Marburg virus nanobodies (VHH or single-domain antibody [sdAb]) targeted a cryptotope within an alpha-helical assembly at the nucleoprotein (NP) C-terminus that was conserved through half a century of viral evolution. Here, we wished to determine whether an anti–Ebola virus sdAb, that was cross-reactive within the Ebolavirus genus, recognized a similar structural feature upstream of the ebolavirus NP C-terminus. In addition, we sought to determine whether the specificities of a less cross-reactive anti–Zaire ebolavirus sdAb and a totally specific anti–Sudan ebolavirus sdAb were the result of exclusion from this region. Binding and X-ray crystallographic studies revealed that the primary determinant of cross-reactivity did indeed appear to be a preference for the helical feature. Specificity, in the case of the Zaire ebolavirus–specific sdAb, arose from the footprint shifting away from the helices to engage more variable residues. While both sdAbs used CDRs, they also had atypical side-on approaches, with framework 2 helping to accommodate parts of the epitope in sizeable paratope gullies. The Sudan ebolavirus–specific sdAb was more remarkable and appeared to bind two C-terminal domains simultaneously via nonoverlapping epitopes—“paratope duality.” One mode involved paratope gullying, whereas the other involved only CDRs, with CDR3 restructuring to wedge in between opposing walls of an interdomain crevice. The varied routes used by sdAbs to engage antigens discovered here deepen our appreciation of the small scaffold's architectural versatility and also reveal lucrative opportunities within the ebolavirus NP C-termini that might be leveraged for diagnostics and novel therapeutic targeting.

Amyloid light chain (AL) amyloidosis is characterized by misfolded light chain (LC) (amyloid) deposition in various peripheral organs, leading to progressive dysfunction and death. There are no regulatory agency–approved treatments for AL amyloidosis, and none of the available standard of care approaches directly targets the LC protein that constitutes the amyloid. NEOD001, currently in late-stage clinical trials, is a conformation-specific, anti-LC antibody designed to specifically target misfolded LC aggregates and promote phagocytic clearance of AL amyloid deposits. The present study demonstrated that the monoclonal antibody 2A4, the murine form of NEOD001, binds to patient-derived soluble and insoluble LC aggregates and induces phagocytic clearance of AL amyloid in vitro. 2A4 specifically labeled all 21 fresh-frozen organ samples studied, which were derived from 10 patients representing both κ and λ LC amyloidosis subtypes. 2A4 immunoreactivity largely overlapped with thioflavin T–positive labeling, and 2A4 bound both soluble and insoluble LC aggregates extracted from patient tissue. Finally, 2A4 induced macrophage engagement and phagocytic clearance of AL amyloid deposits in vitro. These findings provide further evidence that 2A4/NEOD001 can effectively clear and remove human AL-amyloid from tissue and further support the rationale for the evaluation of NEOD001 in patients with AL amyloidosis.

Introduction: Transthyretin amyloidosis (ATTR amyloidosis) is caused by the misfolding and deposition of the transthyretin (TTR) protein and results in progressive multi-organ dysfunction. TTR epitopes exposed by dissociation and misfolding are targets for immunotherapeutic antibodies. We developed and characterized antibodies that selectively bound to misfolded, non-native conformations of TTR. Methods: Antibody clones were generated by immunizing mice with an antigenic peptide comprising a cryptotope within the TTR sequence and screened for specific binding to non-native TTR conformations, suppression of in vitro TTR fibrillogenesis, promotion of antibody-dependent phagocytic uptake of mis-folded TTR and specific immunolabeling of ATTR amyloidosis patient-derived tissue. Results: Four identified monoclonal antibodies were characterized. These antibodies selectively bound the target epitope on monomeric and non-native misfolded forms of TTR and strongly suppressed TTR fibril formation in vitro. These antibodies bound fluorescently tagged aggregated TTR, targeting it for phagocytic uptake by macrophage THP-1 cells, and amyloid-positive TTR deposits in heart tissue from patients with ATTR amyloidosis, but did not bind to other types of amyloid deposits or normal tissue. Conclusions: Conformation-specific anti-TTR antibodies selectively bind amyloidogenic but not native TTR. These novel antibodies may be therapeutically useful in preventing deposition and promoting clearance of TTR amyloid and in diagnosing TTR amyloidosis.

Marburg virus (MARV) is a highly lethal hemorrhagic fever virus that is increasingly re-emerging in Africa, has been imported to both Europe and the US, and is also a Tier 1 bioterror threat. As a negative sense RNA virus, MARV has error prone replication which can yield progeny capable of evading countermeasures. To evaluate this vulnerability, we sought to determine the epitopes of 4 llama single-domain antibodies (sdAbs or VHH) specific for nucleoprotein (NP), each capable of forming MARV monoclonal affinity reagent sandwich assays. Here, we show that all sdAb bound the C-terminal region of NP, which was produced recombinantly to derive X-ray crystal structures of the three best performing antibody-antigen complexes. The common epitope is a trio of alpha helices that form a novel asymmetric basin-like depression that accommodates each sdAb paratope via substantial complementarity-determining region (CDR) restructuring. Shared core contacts were complemented by unique accessory contacts on the sides and overlooks of the basin yielding very different approach routes for each sdAb to bind the antigen. The C-terminal region of MARV NP was unable to be crystallized alone and required engagement with sdAb to form crystals suggesting the antibodies acted as crystallization chaperones. While gross structural homology is apparent between the two most conserved helices of MARV and Ebolavirus, the positions and morphologies of the resulting basins were markedly different. Naturally occurring amino acid variations occurring in bat and human Marburgvirus strains all mapped to surfaces distant from the predicted sdAb contacts suggesting a vital role for the NP interface in virus replication. As an essential internal structural component potentially interfacing with a partner protein it is likely the C-terminal epitope remains hidden or “cryptic” until virion disruption occurs. Conservation of this epitope over 50 years of Marburgvirus evolution should make these sdAb useful foundations for diagnostics and therapeutics resistant to drift.

Summary Five monoclonal antibodies (MAbs) were prepared to particles of potato virus A (PVA), isolate B11. In immunoblots, MAbs A1D8 and A5B6 reacted only with full length molecules of PVA coat protein (CP). Pepscan tests with overlapping octapeptides representing the whole sequence of PVA CP showed that the epitope detected by MAb A5B6 is contained in its N-terminal octapeptide. MAbs A9A4, A3H4 and A6B8 reacted with CP molecules that lacked about 5 kD of sequence at their end(s) and detected epitopes at residues 52 to 62, 64 to 73 and 75 to 82 respectively, all of which lie in the protease-resistant core of the CP. The epitope which reacts with MAb A3H4 is in a region predicted to be hydrophobic and is not detected in intact virus particles, indicating it is a cryptotope. In contrast, MAbs A6B8 and A9A4 reacted with freshly purified PVA particles but more strongly with partially degraded ones. Pepscan tests with polyclonal antibodies to PVA isolate B11 identified five additional immunogenic sequences in PVA CP and showed that regions at the N-termini of the intact and core molecules are immunodominant. PVA isolate B11 was not transmitted by aphids, and its CP N-terminal octapeptide contains the sequence DAS, which is associated with aphid-non-transmissibility in other potyviruses. MAb A5B6, which detects this region, reacted strongly in ELISA with three out of four other aphid-non-transmissible PVA isolates but only weakly with three aphid-transmissible ones, suggesting that differences in N-terminal sequence may underlie most of the differences in aphid transmissibility.

Summary Four monoclonal antibodies (MCA) were obtained to the ‘Ahlum’ serotype of beet soil-borne virus (BSBV). On ELISA plates which had been precoated with polyclonal antibodies (PCA) all four MCA detected this serotype with a higher sensitivity than alkaline phosphatase-labelled PCA. Three of the MCA were specific for the ‘Ahlum’ serotype, but a fourth one also detected the distantly related ‘Wierthe’ serotype. Cross-reactions with wheat soil-borne or oat golden stripe furoviruses were not observed. One of the MCA reacted with an epitope which is exposed along the entire length of the BSBV particles, whereas two others were specific for epitopes which are exposed on one particle extremity only. Although these latter two epitopes occur apparently on the same extremity of the particles, they seem to be different, because one is found only on the particles of the ‘Ahlum’ serotype, whereas the other one is present also on the particles of the ‘Wierthe’ serotype. The fourth MCA is specific for a cryptotope which is not exposed on the intact virus particles, but presumably on some degradation product or precursor of the viral coat protein present in crude sap preparations. All four epitopes are SDS-labile; they are not detected on denatured viral coat protein on Western blots.

The antigenicity of proteins resides in different types of antigenic determinants known as continuous and discontinuous epitopes, cryptotopes, neotopes, and mimotopes. All epitopes have fuzzy boundaries and can be identified only by their ability to bind to certain antibodies. Antigenic cross-reactivity is a common phenomenon because antibodies are always able to recognize a considerable number of related epitopes. This places severe limits to the specificity of antibodies. Antigenicity, which is the ability of an epitope to react with an antibody, must be distinguished from its immunogenicity or ability to induce antibodies in a competent vertebrate host. Failure to make this distinction partly explains why no successful peptide-based vaccines have yet been developed. Methods for predicting the epitopes of proteins are discussed and the reasons for the low success rate of epitope prediction are analyzed.

The role played by studies of tobacco mosaic virus (TMV) in the development of virology is summarized. TMV was the first virus shown to be able to pass through a bacteria-retaining filter; it was also the first virus to be crystallized, to have its morphology and structure elucidated and its coat protein sequenced. Experiments done with TMV RNA in the 1950s established that viral nucleic acid is the carrier of viral infectivity. The mechanism of self-assembly and disassembly of TMV particles is described and the antigenic properties of virions and dissociated coat protein subunits are discussed. Studies of TMV have also led to various biotechnological applications which are briefly described.

Antigenicity or antigenic reactivity refers to the capacity of viruses to bind to specific antibody molecules. The antigenicity of nonenveloped viruses resides in the antigenic sites or B-cell epitopes of capsid proteins that are recognized by the binding sites of antibodies. Protein epitopes are classified as either continuous or discontinuous depending on whether the amino acids included in the epitope are contiguous in the polypeptide chain or not. Most epitopes are discontinuous and since they consist of surface residues brought together by the folding of the peptide chain, their antigenic reactivity depends on the native conformation of the protein. The quaternary structure of viral capsids gives rise to epitopes known as neotopes. Neotopes arise either through conformational changes in the capsid proteins induced by intersubunit interactions or by the juxtaposition of residues from neighboring subunits forming a novel epitope. Immunogenicity is the ability of a protein to give rise to an immune response in a competent host and it can be defined only in the biological context of the host. Knowledge of the viral antigenic sites recognized by antibodies does not necessarily indicate which immunogenic structure initiated the production of antibodies in the immunized host. Failure to differentiate between antigenicity and immunogenicity is responsible for the lack of success in developing synthetic peptide vaccines against viral diseases.

Thirteen hybridoma cell lines secreting monoclonal antibodies (MCAs) to watermelon mosaic virus 2 (WMV-2) were produced by fusing spleen cells of immunized mice and mouse myeloma cell line. The MCAs recognized at least three different epitopes on the particles of WMV-2. WMV-2 and zucchini yellow mosaic virus (ZYMV) shared one epitope (cryptotope) because a group of MCAs (group 3) to WMV-2 reacted only with ZYMV treated using carbonate buffer, pH 9.6 in non-precoated indirect enzyme-linked immunosorbent assay (I-ELISA) but not in precoated I-ELISA with rabbit antiserum to WMV-2. This fact provided a basis for clarification of the serological relationship between the two viruses. ELISA using MCA WMV-2 15 was found to be useful for WMV-2 detection and/or diagnosis of field samples.

We describe the expression of the bispecific antibody biscFv2429 in transgenic suspension culture cells and tobacco plants. biscFv2429 consists of two single-chain antibodies, scFv24 and scFv29, connected by the Trichoderma reesi cellobiohydrolase I linker. biscFv2429 binds two epitopes of tobacco mosaic virus (TMV): the scFv24 domain recognizes neotopes of intact virions, and the scFv29 domain recognizes a cryptotope of the TMV coat protein monomer. biscFv2429 was functionally expressed either in the cytosol (biscFv2429-cyt) or targeted to the apoplast using a murine leader peptide sequence (biscFv2429-apoplast). A third construct contained the C-terminal KDEL sequence for retention in the ER (biscFv2429-KDEL). Levels of cytoplasmic biscFv2429 expression levels were low. The highest levels of antibody expression were for apoplast-targeted biscFv2429-apoplast and ER-retained biscFv2429-KDEL that reached a maximum expression level of 1.65% total soluble protein in transgenic plants. Plant-expressed biscFv2429 retained both epitope specificities, and bispecificity and bivalency were confirmed by ELISA and surface plasmon resonance analysis. This study establishes plant cells as an expression system for bispecific single-chain antibodies for use in medical and biological applications.

A panel of 15 mouse monoclonal antibodies (MAbs) was raised against five strains of alfalfa mosaic virus (AMV) which were closely related antigenically but biologically distinct. A wide diversity of MAb specificity was revealed by screening them in three formats of indirect ELISA, using native and glutaraldehyde-fixed AMV particles as well as isolated coat protein preparations. Of these MAbs, seven reacted specifically with only one AMV strain in at least one ELISA format and at least one MAb was capable of identifying each of the strains. One of the MAbs reacted with a cryptotope, whereas the other recognized different subtypes of either metatopes or neotopes, indicating that the AMV particle has a complex antigenic structure. Only two of the MAbs precipitated AMV in agarose gels. Another two, which recognized epitopes on coat protein subunits, also reacted well in immunoblots. One of the precipitating MAbs recognized an epitope which appears to be common to AMV and cucumber mosaic virus.