Serum immunoglobulin or albumin binding single-domain antibodies that enable tailored half-life extension of biologics in multiple animal species.

Single-domain antibody fragments (sdAbs) can be isolated from heavy-chain-only antibodies that occur in camelids or the heavy chain of conventional antibodies, that also occur in camelids. Therapeutic application of sdAbs is often complicated by their low serum half-life. Fusion to sdAb that bind to long-lived serum proteins albumin or IgG can prolong serum half-life of fusion partners. Such studies mostly focused on human application. For half-life prolongation in multiple animal species novel species cross-reacting sdAb are needed. We here describe the isolation from immunized llamas of sdAbs G6 and G13 that bound IgG of 9-10 species analysed, including horse, dog, cat, and swine, as well as sdAb A12 that bound horse, dog, swine and cat albumin. A12 bound albumin with 13 to 271 nM affinity dependent on the species. G13 affinity was difficult to determine by biolayer interferometry due to low and heterogeneous signals. G13 and G6 compete for the same binding domain on Fab fragments. Furthermore, they both lack the hallmark residues typical of camelid sdAbs derived from heavy-chain antibodies and had sequence characteristics typical of human sdAbs with high solubility and stability. This suggests they are derived from conventional llama antibodies. They most likely bind IgG through pairing with VL domains at the VH-VL interface rather than a paratope involving complementarity determining regions. None of the isolated sdAb interfered with FcRn binding to albumin or IgG, and thus do not prevent endosomal albumin/IgG-sdAb complex recycling. Fusions of albumin-binding sdAb A12 to several tetanus neurotoxin (TeNT) binding sdAbs prolonged the terminal serum half-life in piglets to about 4 days, comparable to authentic swine albumin. However, G13 conferred a much lower half-life of 0.84 days. Similarly, in horse, G13 prolonged half-life to only 1.2 days whereas A12 fused to two TeNT binding domains (T6T16A12) had a half-life of 21 days. The high half-life of T6T16A12, which earlier proved to be a highly potent TeNT antitoxin, further supports its therapeutic value. Furthermore, we have identified several additional sdAbs that enable tailored half-life extension of biologicals in multiple animal species.

Single-Domain Antibody Multimers for Detection of Botulinum Neurotoxin Serotypes C, D, and Their Mosaics in Endopep-MS.

Botulinum neurotoxins (BoNTs) are highly toxic proteins that require high-affinity immunocapture reagents for use in endopeptidase-based assays. Here, 30 novel and 2 earlier published llama single-domain antibodies (VHHs) against the veterinary-relevant BoNT serotypes C and D were yeast-produced. These VHHs recognized 10 independent antigenic sites, and many cross-reacted with the BoNT/DC and CD mosaic variants. As VHHs are highly suitable for genetically linking to increase antigen-binding affinity, 52 VHH multimers were produced and their affinity for BoNT/C, D, DC, and CD was determined. A selection of 15 multimers with high affinity (KD < 0.1 nM) was further shown to be resilient to a high salt wash that is used for samples from complex matrices and bound native BoNTs from culture supernatants as shown by Endopep-MS. High-affinity multimers suitable for further development of a highly sensitive Endopep-MS assay include four multimers that bind both BoNT/D and CD with KD of 14-99 pM, one multimer for BoNT/DC (65 pM) that also binds BoNT/C (75 pM), and seven multimers for BoNT/C (<1-19 pM), six of which also bind BoNT/DC with lower affinity (93-508 pM). In addition to application in diagnostic tests, these VHHs could be used for the development of novel therapeutics for animals or humans.

Small-Scale Secretory VHH Expression in Saccharomyces cerevisiae.

After isolation of a single-domain antibody (VHH) binding to an antigen of interest, the soluble VHH is often produced in Escherichia coli. However, targeting VHH expression to the secretory pathway of Saccharomyces cerevisiae (baker's yeast) enables the secretion of correctly folded, soluble, disulfide-bonded, and N-glycosylated VHHs into the culture medium. Here, we describe the small-scale production of VHHs in baker's yeast in shaker flasks using both an episomal vector and a vector requiring genomic integration for higher VHH expression levels. This expression system results in the production of VHHs linked to the natural llama long hinge region including a single cysteine residue for partial dimerization. This format is especially suitable for the development of double antibody sandwich ELISAs by passive adsorption of unlabeled VHHs to polystyrene ELISA plates, antigen capture, and detection of the antigen of interest using a second biotinylated VHH. The procedures described here for detection of foot-and-mouth disease virus can also be applied to other antigens for which suitable VHHs are available.

Mapping of foot-and-mouth disease virus antigenic sites recognized by single-domain antibodies reveals different 146S particle specific sites and particle flexibility.

Vaccination with intact (146S) foot-and-mouth disease virus (FMDV) particles is used to control FMD. However, 146S particles easily dissociate into stable pentameric 12S particles which are less immunogenic. We earlier isolated several single-domain antibody fragments (VHHs) that specifically bind either 146S or 12S particles. These particle-specific VHHs are excellent tools for vaccine quality control. In this study we mapped the antigenic sites recognized by these VHHs by competition ELISAs, virus neutralization, and trypsin sensitivity of epitopes. We included two previously described monoclonal antibodies (mAbs) that are either 12S specific (mAb 13A6) or 146S specific (mAb 9). Although both are 12S specific, the VHH M3F and mAb 13A6 were found to bind independent antigenic sites. M3F recognized a non-neutralizing and trypsin insensitive site whereas mAb 13A6 recognized the trypsin sensitive VP2 N-terminus. The Asia1 146S-specific site was trypsin sensitive, neutralizing and also recognized by the VHH M8F, suggesting it involves the VP1 GH-loop. The type A 146S-specific VHHs recognized two independent antigenic sites that are both also neutralizing but trypsin insensitive. The major site was further mapped by cross-linking mass spectrometry (XL-MS) of two broadly strain reactive 146S-specific VHHs complexed to FMDV. The epitopes were located close to the 2-fold and 3-fold symmetry axes of the icosahedral virus 3D structure, mainly on VP2 and VP3, overlapping the earlier identified mAb 9 site. Since the epitopes were located on a single 12S pentamer, the 146S specificity cannot be explained by the epitope being split due to 12S pentamer dissociation. In an earlier study the cryo-EM structure of the 146S-specific VHH M170 complexed to type O FMDV was resolved. The 146S specificity was reported to be caused by an altered conformation of this epitope in 12S and 146S particles. This mechanism probably also explains the 146S-specific binding by the two type A VHHs mapped by XL-MS since their epitopes overlapped with the epitope recognized by M170. Surprisingly, residues internal in the 146S quaternary structure were also cross-linked to VHH. This probably reflects particle flexibility in solution. Molecular studies of virus-antibody interactions help to further optimize vaccines and improve their quality control.

Novel Capsid-Specific Single-Domain Antibodies with Broad Foot-and-Mouth Disease Strain Recognition Reveal Differences in Antigenicity of Virions, Empty Capsids, and Virus-Like Particles.

Foot-and-mouth disease (FMD) vaccine efficacy is mainly determined by the content of intact virions (146S) and empty capsids (75S). Both particles may dissociate into 12S subunits upon vaccine manufacturing, formulation, and storage, reducing vaccine potency. We report the isolation of capsid-specific llama single-domain antibodies (VHHs) with broad strain recognition that can be used to quantify intact capsids in FMD vaccines by double antibody sandwich (DAS) ELISA. One capsid-specific VHH displayed remarkably broad strain reactivity, recognizing 14 strains representing the 13 most important lineages of serotype A, and two VHHs cross-reacted with other serotypes. We additionally show that the newly isolated VHHs, as well as previously characterized VHHs, can be used to identify antigenic differences between authentic 146S and 75S capsids, as well as corresponding genetically engineered virus-like particles (VLPs). Our work underscores that VHHs are excellent tools for monitoring the quantity and stability of intact capsids during vaccine manufacturing, formulation, and storage, and additionally shows that VHHs can be used to predict the native-like structure of VLPs.

A novel single-domain antibody multimer that potently neutralizes tetanus neurotoxin.

Tetanus antitoxin, produced in animals, has been used for the prevention and treatment of tetanus for more than 100 years. The availability of antitoxins, ethical issues around production, and risks involved in the use of animal derived serum products are a concern. We therefore developed a llama derived single-domain antibody (VHH) multimer to potentially replace the conventional veterinary product. In total, 28 different tetanus neurotoxin (TeNT) binding VHHs were isolated, 14 of which were expressed in yeast for further characterization. Four VHH monomers (T2, T6, T15 and T16) binding TeNT with high affinity (KD < 1 nM), covering different antigenic domains as revealed by epitope binning, and including 3 monomers (T6, T15 and T16) that inhibited TeNT binding to neuron gangliosides, were chosen as building blocks to generate 11 VHH multimers. These multimers contained either 1 or 2 different TeNT binding VHHs fused to 1 VHH binding to either albumin (A12) or immunoglobulin (G13) to extend serum half-life in animals. Multimers consisting of 2 TeNT binding VHHs showed more than a 10-fold increase in affinity (KD of 4-23 pM) when compared to multimers containing only one TeNT binding VHH. The T6 and T16 VHHs showed synergistic in vivo TeNT neutralization and, when incorporated into a single VHH trimer (T6T16A12), they showed a very high TeNT neutralizing capacity (1,510 IU/mg).

Chimeric O1K foot-and-mouth disease virus with SAT2 outer capsid as an FMD vaccine candidate.

Foot-and-mouth disease virus (FMDV) is highly contagious and infects cloven-hoofed domestic livestock leading to foot-and-mouth disease (FMD). FMD outbreaks have severe economic impact due to production losses and associated control measures. FMDV is found as seven distinct serotypes, but there are numerous subtypes within each serotype, and effective vaccines must match the subtypes circulating in the field. In addition, the O and Southern African Territories (SAT) serotypes, are relatively more thermolabile and their viral capsids readily dissociate into non-immunogenic pentameric subunits, which can compromise the effectiveness of FMD vaccines. Here we report the construction of a chimeric clone between the SAT2 and O serotypes, designed to have SAT2 antigenicity. Characterisation of the chimeric virus showed growth kinetics equal to that of the wild type SAT2 virus with better thermostability, attributable to changes in the VP4 structural protein. Sequence and structural analyses confirmed that no changes from SAT2 were present elsewhere in the capsid as a consequence of the VP4 changes. Following exposure to an elevated temperature the thermostable SAT2-O1K chimera induced higher neutralizing-antibody titres in comparison to wild type SAT2 virus.

Isolation of Single-Domain Antibody Fragments That Preferentially Detect Intact (146S) Particles of Foot-and-Mouth Disease Virus for Use in Vaccine Quality Control.

Intact (146S) foot-and-mouth disease virus (FMDVs) can dissociate into specific (12S) viral capsid degradation products. FMD vaccines normally consist of inactivated virions. Vaccine quality is dependent on 146S virus particles rather than 12S particles. We earlier isolated two llama single-domain antibody fragments (VHHs) that specifically recognize 146S particles of FMDV strain O1 Manisa and shown their potential use in quality control of FMD vaccines during manufacturing. These 146S-specific VHHs were specific for particular O serotype strains and did not bind strains from other FMDV serotypes. Here, we describe the isolation of 146S-specific VHHs against FMDV SAT2 and Asia 1 strains by phage display selection from llama immune libraries. VHHs that bind both 12S and 146S particles were readily isolated but VHHs that bind specifically to 146S particles could only be isolated by phage display selection using prior depletion for 12S particles. We obtained one 146S-specific VHH-M332F-that binds to strain Asia 1 Shamir and several VHHs that preferentially bind 146S particles of SAT2 strain SAU/2/00, from which we selected VHH M379F for further characterization. Both M332F and M379F did not bind FMDV strains from other serotypes. In a sandwich enzyme-linked immunosorbent assay (ELISA) employing unlabeled and biotinylated versions of the same VHH M332F showed high specificity for 146S particles but M379F showed lower 146S-specificity with some cross-reaction with 12S particles. These ELISAs could detect 146S particle concentrations as low as 2.3-4.6 µg/l. They can be used for FMD vaccine quality control and research and development, for example, to identify virion stabilizing excipients.

Identification of the structural basis of thermal lability of a virus provides a rationale for improved vaccines.

Virus stability and dynamics play critical roles during infection. Some viruses, including foot-and-mouth disease virus (FMDV), are surprisingly prone to thermal dissociation outside the cell. The structural bases and functional implications of this distinctive trait were essentially unknown. This study (1) uncovers the structural determinants of FMDV thermolability, (2) investigates the relationship between virus thermolability and infectivity, and (3) provides a structure-based rationale for engineering thermostable virus particles to develop improved vaccines and nanocontainers. The results reveal that negatively charged residues close to protein-protein interfaces exert electrostatic repulsions between capsid subunits and mediate the sensitivity of the virion to thermal dissociation, even at neutral pH. Based on these results, a series of fully infectious virions of increased thermostability were engineered by individually removing different carboxylates involved in intersubunit repulsions. The implications for virus biology and the design of thermostable vaccines are discussed.

The effect of uniform capture molecule orientation on biosensor sensitivity: dependence on analyte properties.

Uniform orientation of capture molecules on biosensors has been reported to increase sensitivity. Here it is investigated which analyte properties contribute to sensitivity by orientation. Orientation of capture molecules on biosensors was investigated using variable domains of llama heavy-chain antibodies (VHHs) as capture molecule, and a surface plasmon resonance (SPR) chip as biosensor. Two VHHs were tested in this study: one recognizing foot-and-mouth disease virus (FMDV) and another recognizing the 16 kDa heat-shock protein of Mycobacterium tuberculosis. SPR chips with randomly immobilized biotinylated VHHs were compared to streptavidin-coated SPR chips, on which similar quantities of oriented biotinylated VHHs were non-covalently immobilized. Analytes that differ in molecular weight, epitope number and epitope affinity were compared using the FMDV-recognizing VHH. When binding of intact FMDV particles (146 S; 8200 kDa) or pentameric FMDV coat protein aggregates (12 S; 282 kDa) was detected, a modest (1-2-fold) increase in sensitivity was observed. When a 26-residue peptide (3 kDa) containing the epitope for VHH recognition was tested, much larger effects of capture molecule orientation (14-fold) on signal were observed. A 20-227-fold improvement was also observed when the epitope peptide was covalently linked to bovine serum albumin (67 kDa) or R-phycoerythrin (240 kDa). The results indicate that orientation of the capture molecule hardly affects high-affinity interactions, while it leads to strong improvements in sensitivity for lower-affinity interactions.

Characterization of epitope-tagged foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals with an almost-worldwide distribution. Conventional FMD vaccines consisting of chemically inactivated viruses have aided in the eradication of FMD from Europe and remain the main tool for control in endemic countries. Although significant steps have been made to improve the quality of vaccines, such as improved methods of antigen concentration and purification, manufacturing processes are technically demanding and expensive. Consequently, there is large variation in the quality of vaccines distributed in FMD-endemic countries compared with those manufactured for emergency use in FMD-free countries. Here, we have used reverse genetics to introduce haemagglutinin (HA) and FLAG tags into the foot-and-mouth disease virus (FMDV) capsid. HA- and FLAG-tagged FMDVs were infectious, with a plaque morphology similar to the non-tagged parental infectious copy virus and the field virus. The tagged viruses utilized integrin-mediated cell entry and retained the tag epitopes over serial passages. In addition, infectious HA- and FLAG-tagged FMDVs were readily purified from small-scale cultures using commercial antibodies. Tagged FMDV offers a feasible alternative to the current methods of vaccine concentration and purification, a potential to develop FMD vaccine conjugates and a unique tool for FMDV research.

Improved functional immobilization of llama single-domain antibody fragments to polystyrene surfaces using small peptides.

We studied the effect of different fusion domains on the functional immobilization of three llama single-domain antibody fragments (VHHs) after passive adsorption to polystyrene in enzyme-linked immunosorbent assays (ELISA). Three VHHs produced without any fusion domain were efficiently adsorbed to polystyrene, which, however, resulted in inefficient antigen binding. Functional VHH immobilization was improved by VHH fusion to a consecutive myc-His6-tag and was even more improved by fusion to the llama antibody long hinge region containing an additional His6-tag (LHc-His6). The partial dimerization of VHH-LHc-His6 fusion proteins through LHc-mediated disulfide-bond formation was not essential for their improved functional immobilization. VHH fusions to specific polystyrene binding peptides, hydrophobins, or other, unrelated VHH domains were less suitable for increasing functional VHH immobilization because of reduced microbial expression levels. Thus, VHH-LHc-His6 fusion proteins are most suited for functional passive adsorption in ELISA.

Parenteral vaccination of mammalian livestock with Newcastle disease virus-based vector vaccines offers optimal efficacy and safety.

Newcastle disease virus (NDV) is an avian virus that is being evaluated as a vaccine vector for the delivery of foreign genes in mammals. The use of NDV as a vaccine vector in these species offers two major advantages. First, NDV is highly attenuated in mammals, rendering its use inherently safe. Second, mammals lack pre-existing NDV immunity, which minimizes the risk of vaccination failure. NDV-vector vaccines are generally administered to mammals via the respiratory route. We recently showed that intramuscular vaccination with NDV-based Rift Valley fever virus (RVFV) vaccines provides complete protection in mice and induces neutralizing antibodies in sheep and cattle, the main target species of RVFV. Here, we discuss the use of NDV as a vaccine vector for applications in mammalian livestock with an emphasis on the vaccination route. We also report the results of novel experiments that underscore our notion that vaccination via a parenteral route is more effective than immunization via the respiratory route.

Prolonged in vivo residence times of llama single-domain antibody fragments in pigs by binding to porcine immunoglobulins.

The therapeutic parenteral application of llama single-domain antibody fragments (VHHs) is hampered by their small size, resulting in a fast elimination from the body. Here we describe a method to increase the serum half-life of VHHs in pigs by fusion to another VHH binding to porcine immunoglobulin G (pIgG). We isolated 19 pIgG-binding VHHs from an immunized llama using phage display. Six VHHs were genetically fused to model VHH K 609 that binds to Escherichia coli F4 fimbriae. All six yeast-produced genetic fusions of two VHH domains (VHH2s) were functional in ELISA and bound to pIgG with high affinity (1-33 nM). Four pIgG-binding VHH2s were administered to pigs and showed a 100-fold extended in vivo residence times as compared to a control VHH2 that does not bind to pIgG. This could provide the basis for therapeutic application of VHHs in pigs.

Fasciola hepatica procathepsin L3 protein expressed by a baculovirus recombinant can partly protect rats against fasciolosis.

Fasciola hepatica juveniles express immunodominant cathepsin L proteins, which are mainly found in their immature, procathepsin form. A gene encoding such a procathepsin L (FheCL3) was expressed by a baculovirus recombinant and by Saccharomyces cerevisiae. The glycosylated FheCL3 proteins obtained by both systems were used in a vaccination/challenge experiment in rats. Both antigens evoked similar antibody responses, but only the baculovirus expressed FheCL3 caused a significant protection against the number of liver flukes (52% protection, P=0.01), whereas the S. cerevisiae expressed FheCL3 did not. In a second experiment in rats, deglycosylated versions of both antigens were used, but this did not improve their efficacies.

Identification of a novel Fasciola hepatica cathepsin L protease containing protective epitopes within the propeptide.

Cathepsin L (CL)-like proteases are important candidate vaccine antigens for protection against helminth infections. We previously identified an immunogenic 32 kDa protein specifically present in newly excysted juveniles (NEJs) of Fasciola hepatica. Here we show by N-terminal protein sequencing that this protein represents a CL-like protease still containing the propeptide. Two cDNAs encoding this CL were subsequently isolated from NEJs by RT-PCR. The predicted amino acid sequences of these cDNAs showed approximately 70% sequence homology to both CL1 and CL2 sequences isolated from adult stage F. hepatica and are, therefore, referred to as CL3. The CL3 clones encoded asparagine at position P1 of the propeptide cleavage site, suggesting a dependence on asparaginyl endopeptidases for maturation. Recombinant expression of a CL3 cDNA in Saccharomyces cerevisiae resulted in secretion of the proenzyme form. The propeptide of CL-like proteins was predicted to contain important B-cell epitopes. To determine the contribution of the propeptide to protective immunity, rats were vaccinated with Keyhole Limpet Haemocyanin-conjugated synthetic peptides encoding these putative B-cell epitopes derived from the CL1 or CL3 sequence. A subsequent challenge infection resulted in a significant (P < 0.05) reduction of fluke load compared to adjuvant controls. We conclude that the propeptide of CL3 plays an important role in inducing immunity against F. hepatica infection.