An innovative method for characterizing neutralizing antibodies against antibody-derived therapeutics.

Detection of anti-drug antibodies (ADA) that have a neutralizing capacity is an important aspect of immunogenicity evaluation during development of biotherapeutics, but developing and validating neutralizing antibody (NAb) assays that show direct interference of a biologic function is a challenging and resource-intensive activity. In particular, the need for adequate drug and target tolerance often requires extensive pre-treatment steps that limit assay sensitivity compared with a typical bridging-format assay used to detect binding ADA. Such limitations may complicate data interpretation as a positive ADA followed by a negative NAb result could be due to the presence of non-neutralizing antibodies or could be a false-negative for NAbs due to methodology differences. To address such issues, we developed a novel assay for Nanobodies® and other antibody-derived therapeutics that solely detects ADA directed against the complementarity-determining regions (CDRs) involved in drug-target interactions. This was achieved by creating a "null variant" of the therapeutic drug, which has mutated CDRs rendering it non-functional for target binding but is otherwise identical to the drug compound. Non-CDR-binding antibodies are pre-complexed with the null variant of the Nanobody leaving only CDR-binding ADA with neutralizing potential (ANP) to be detected in this assay, which is called a NAb Epitope Characterization Assay (NECA). Method qualification results confirmed highly comparable assay characteristics (sensitivity, drug tolerance, selectivity and precision) of both the NECA and a validated ADA assay for the same Nanobody. A panel of purified neutralizing and non-neutralizing antibodies as well as non-clinical and clinical samples were used to further substantiate the fit-for-purpose and advantages of this novel assay format to detect ANP. In the clinical case study, a 20 to 40-fold difference in assay sensitivity existed between the validated ADA assay and NAb assay, which complicated data interpretation. Implementation of the NECA allowed unambiguous comparison of the levels of binding ADA and ANP in study samples which enabled us to delineate the true neutralizing capacity of the responses. Depending on the risk of the therapeutic, this method could be a valuable alternative for NAb testing by enabling earlier detection of ADA with neutralizing potential and ensuring adequate immunogenicity risk assessment.

Workshop on Recent Issues in Bioanalysis (WRIB) Poster Award winners 2017.

The 11th WRIB held in Los Angeles, CA, USA in April 2017. It drew over 750 professionals representing large Pharmas, Biotechs, CROs and multiple regulatory agencies from around the world, from the global bioanalytical community. Bioanalysis and Bioanalysis Zone are very proud to be supporting the WRIB Poster Awards again this year, and we feature the profiles of the authors of the winning posters. Visit www.bioanalysis-zone.com to see the winning posters in full.

Cranberry extract inhibits in vitro adhesion of F4 and F18+Escherichia coli to pig intestinal epithelium and reduces in vivo excretion of pigs orally challenged with F18+ verotoxigenic E. coli.

F4+E. coli and F18+E. coli infections are an important threat for pig industry worldwide. Antibiotics are commonly used to treat infected piglets, but the emerging development of resistance against antibiotics raises major concerns. Hence, alternative therapies to prevent pigs from F4+E. coli and F18+E. coli infections need to be developed. Since cranberry previously showed anti-adhesive activity against uropathogenic E. coli, we aimed to investigate whether cranberry extract could also inhibit binding of F4+E. coli and F18+E. coli to pig intestinal epithelium. Using the in vitro villus adhesion assay, we found that low concentrations of cranberry extract (20µg or 100µg/ml) have strong inhibitory activity on F4+E. coli (75.3%, S.D.=9.31 or 95.8%, S.D.=2.56, respectively) and F18+E. coli adherence (100% inhibition). This effect was not due to antimicrobial activity. Moreover, cranberry extract (10mg or 100mg) could also abolish in vivo binding of F4 and F18 fimbriae to the pig intestinal epithelium in ligated loop experiments. Finally, two challenge experiments with F18+E. coli were performed to address the efficacy of in-feed or water supplemented cranberry extract. No effect could be observed in piglets that received cranberry extract only in feed (1g/kg or 10g/kg). However, supplementation of feed (10g/kg) and drinking water (1g/L) significantly decreased excretion and diarrhea. The decreased infection resulted in a decreased serum antibody response indicating reduced exposure to F18+E. coli.

High susceptibility prevalence for F4+ and F18+Escherichia coli in Flemish pigs.

F4 and/or F18 enterotoxigenic Escherichia coli (F4+/F18+ ETEC) are responsible for diarrhea while F18+ verotoxigenic E. coli (F18+ VTEC) cause edema disease in pigs. Both infections can result in severe economic losses, which are mainly the result of the medication, growth retardation and mortality. The susceptibility of piglets to these pathogens is determined by the presence of F4 and F18 receptors (F4R and F18R). Understanding the composition of the susceptibility phenotypes of pigs is useful for animal health and breeding management. This study aimed to determine the prevalence of the F4 ETEC susceptibility phenotypes and F18+E. coli susceptibility among Flemish pig breeds by using the in vitro villous adhesion assay. In this study, seven F4 ETEC susceptibility phenotypes were found, namely A (F4abR+,acR+,adR+; 59.16%), B (F4abR+,acR+,adR-; 6.28%), C (F4abR+,acR-,adR+; 2.62%), D (F4abR-,acR-,adR+; 6.28%), E (F4abR-,acR-,adR-; 24.08%), F (F4abR+,acR-,adR-; 1.05%) and G (F4abR-,acR+,adR-; 0.52%). F4ab and F4ac E. coli showed a stronger degree of adhesion to the intestinal villi (53.40% and 52.88% strong adhesion, respectively), compared to F4ad E. coli (43.46% strong adhesion). Furthermore, the correlation between F4ac and F4ab adhesion was higher (r=0.78) than between F4ac and F4ad adhesion (r=0.41) and between F4ab and F4ad adhesion (r=0.57). For F18+E. coli susceptibility, seven out of 82 pigs were F18R negative (8.54%), but only two of these seven pigs (2.44%) were also negative for F4R. As such, the chance to identify a pig that is positive for a F4 ETEC variant or F18+E. coli is 97.56%. Therefore, significant economic losses will arise due to F4+ and/or F18+E. coli infections in the Flemish pig population due to the high susceptibility prevalence.

Nanobody mediated inhibition of attachment of F18 Fimbriae expressing Escherichia coli.

Post-weaning diarrhea and edema disease caused by F18 fimbriated E. coli are important diseases in newly weaned piglets and lead to severe production losses in farming industry. Protective treatments against these infections have thus far limited efficacy. In this study we generated nanobodies directed against the lectin domain of the F18 fimbrial adhesin FedF and showed in an in vitro adherence assay that four unique nanobodies inhibit the attachment of F18 fimbriated E. coli bacteria to piglet enterocytes. Crystallization of the FedF lectin domain with the most potent inhibitory nanobodies revealed their mechanism of action. These either competed with the binding of the blood group antigen receptor on the FedF surface or induced a conformational change in which the CDR3 region of the nanobody displaces the D″-E loop adjacent to the binding site. This D″-E loop was previously shown to be required for the interaction between F18 fimbriated bacteria and blood group antigen receptors in a membrane context. This work demonstrates the feasibility of inhibiting the attachment of fimbriated pathogens by employing nanobodies directed against the adhesin domain.

Variation in 12 porcine genes involved in the carbohydrate moiety assembly of glycosphingolipids does not account for differential binding of F4 Escherichia coli and their fimbriae.

BACKGROUND: Glycosphingolipids (GSLs) are important membrane components composed of a carbohydrate structure attached to a hydrophobic ceramide. They can serve as specific membrane receptors for microbes and microbial products, such as F4 Escherichia coli (F4 ETEC) and isolated F4 fimbriae. The aim of this study was to investigate the hypothesis that variation in genes involved in the assembly of the F4 binding carbohydrate moiety of GSLs (i.e. ARSA, B4GALT6, GAL3ST1, GALC, GBA, GLA, GLB1, GLB1L, NEU1, NEU2, UGCG, UGT8) could account for differential binding of F4 ETEC and their fimbriae. RESULTS: RT-PCR could not reveal any differential expression of the 12 genes in the jejunum of F4 receptor-positive (F4R(+)) and F4 receptor-negative (F4R(-)) pigs. Sequencing the complete open reading frame of the 11 expressed genes (NEU2 was not expressed) identified 72 mutations. Although some of them might have a structural effect, none of them could be associated with a F4R phenotype. CONCLUSION: We conclude that no regulatory or structural variation in any of the investigated genes is responsible for the genetic susceptibility of pigs towards F4 ETEC.

Porcine intestinal glycosphingolipids recognized by F6-fimbriated enterotoxigenic Escherichia coli.

One important virulence factor of enterotoxigenic Escherichia coli is their ability to adhere via fimbrial adhesins to specific receptors located on the intestinal mucosa. Here, the potential glycosphingolipid receptors of enterotoxigenic F6-fimbriated E. coli were examined by binding of purified F6 fimbriae, and F6-expressing bacteria, to glycosphingolipids on thin-layer chromatograms. When intestinal mucosal non-acid glycosphingolipids from single pigs were assayed for F6 binding capacity, a selective interaction with two glycosphingolipids was observed. The binding-active glycosphingolipids were isolated and characterized as lactotriaosylceramide (GlcNAcß3Galß4Glcß1Cer) and lactotetraosylceramide (Galß3GlcNAcß3Galß4Glcß1Cer). Further binding assays using a panel of reference glycosphingolipids showed a specific interaction between the F6 fimbriae and a number of neolacto core chain (Galß4GlcNAc) glycosphingolipids. In addition, an occasional binding of the F6 fimbriae to sulfatide, galactosylceramide, lactosylceramide with phytosphingosine and/or hydroxy fatty acids, isoglobotriaosylceramide, gangliotriaosylceramide, and gangliotetraosylceramide was obtained. From the results we conclude that lactotriaosylceramide and lactotetraosylceramide are major porcine intestinal receptors for F6-fimbriated E. coli.

Refined candidate region for F4ab/ac enterotoxigenic Escherichia coli susceptibility situated proximal to MUC13 in pigs.

F4 enterotoxigenic Escherichia coli (F4 ETEC) are an important cause of diarrhea in neonatal and newly-weaned pigs. Based on the predicted differential O-glycosylation patterns of the 2 MUC13 variants (MUC13A and MUC13B) in F4ac ETEC susceptible and F4ac ETEC resistant pigs, the MUC13 gene was recently proposed as the causal gene for F4ac ETEC susceptibility. Because the absence of MUC13 on Western blot from brush border membrane vesicles of F4ab/acR+ pigs and the absence of F4ac attachment to immunoprecipitated MUC13 could not support this hypothesis, a new GWAS study was performed using 52 non-adhesive and 68 strong adhesive pigs for F4ab/ac ETEC originating from 5 Belgian farms. A refined candidate region (chr13: 144,810,100-144,993,222) for F4ab/ac ETEC susceptibility was identified with MUC13 adjacent to the distal part of the region. This candidate region lacks annotated genes and contains a sequence gap based on the sequence of the porcine GenomeBuild 10.2. We hypothesize that a porcine orphan gene or trans-acting element present in the identified candidate region has an effect on the glycosylation of F4 binding proteins and therefore determines the F4ab/ac ETEC susceptibility in pigs.

Expression of verocytotoxic Escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model.

Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and possible adverse effects on the oral administration of obtained tobacco seeds were evaluated in a mouse model. Tobacco was transformed via Agrobacteium tumefaciens with chimeric constructs containing structural parts of the major subunit FedA of the F18 adhesive fimbriae and VT2e B-subunit genes under control of a seed specific GLOB promoter. We showed that the foreign Vt2e-B and F18 genes were stably accumulated in storage tissue by the immunostaining method. In addition, Balb-C mice receiving transgenic tobacco seeds via the oral route showed a significant increase in IgA-positive plasma cell presence in tunica propria when compared to the control group with no observed adverse effects. Our findings encourage future studies focusing on swine for evaluation of the protective effects of transformed tobacco seeds against E. coli infection.

Orally fed seeds producing designer IgAs protect weaned piglets against enterotoxigenic Escherichia coli infection.

Oral feed-based passive immunization can be a promising strategy to prolong maternal lactogenic immunity against postweaning infections. Enterotoxigenic Escherichia coli (ETEC)-caused postweaning diarrhea in piglets is one such infection that may be prevented by oral passive immunization and might avert recurrent economic losses to the pig farming industry. As a proof of principle, we designed anti-ETEC antibodies by fusing variable domains of llama heavy chain-only antibodies (VHHs) against ETEC to the Fc part of a porcine immunoglobulin (IgG or IgA) and expressed them in Arabidopsis thaliana seeds. In this way, four VHH-IgG and four VHH-IgA antibodies were produced to levels of about 3% and 0.2% of seed weight, respectively. Cotransformation of VHH-IgA with the porcine joining chain and secretory component led to the production of light-chain devoid, assembled multivalent dimeric, and secretory IgA-like antibodies. In vitro analysis of all of the antibody-producing seed extracts showed inhibition of bacterial binding to porcine gut villous enterocytes. However, in the piglet feed-challenge experiment, only the piglets receiving feed containing the VHH-IgA-based antibodies (dose 20 mg/d per pig) were protected. Piglets receiving the VHH-IgA-based antibodies in the feed showed a progressive decline in shedding of bacteria, significantly lower immune responses corroborating reduced exposure to the ETEC pathogen, and a significantly higher weight gain compared with the piglets receiving VHH-IgG producing (dose 80 mg/d per pig) or wild-type seeds. These results stress the importance of the antibody format in oral passive immunization and encourage future expression of these antibodies in crop seeds.

Structural insight in histo-blood group binding by the F18 fimbrial adhesin FedF.

F18-positive enterotoxigenic and Shiga toxin-producing Escherichia coli are responsible for post-weaning diarrhoea and oedema disease in pigs and lead to severe production losses in the farming industry. F18 fimbriae attach to the small intestine of young piglets by latching onto glycosphingolipids with A/H blood group determinants on type 1 core. We demonstrate the N-terminal domain of the F18 fimbrial subunit FedF to be responsible for ABH-mediated attachment and present its X-ray structure in ligand-free form and bound to A and B type 1 hexaoses. The FedF lectin domain comprises a 10-stranded immunoglobulin-like ß-sandwich. Three linear motives, Q(47) -N(50), H(88) -S(90) and R(117) -T(119), form a shallow glycan binding pocket near the tip of the domain that is selective for type 1 core glycans in extended conformation. In addition to the glycan binding pocket, a polybasic loop on the membrane proximal surface of FedF lectin domain is shown to be required for binding to piglet enterocytes. Although dispensable for ABH glycan recognition, the polybasic surface adds binding affinity in the context of the host cell membrane, a mechanism that is proposed to direct ABH-glycan binding to cell-bound glycosphingolipids and could allow bacteria to avoid clearance by secreted glycoproteins.

Erythrocyte and porcine intestinal glycosphingolipids recognized by F4 fimbriae of enterotoxigenic Escherichia coli.

Enterotoxigenic F4-fimbriated Escherichia coli is associated with diarrheal disease in neonatal and postweaning pigs. The F4 fimbriae mediate attachment of the bacteria to the pig intestinal epithelium, enabling an efficient delivery of diarrhea-inducing enterotoxins to the target epithelial cells. There are three variants of F4 fimbriae designated F4ab, F4ac and F4ad, respectively, having different antigenic and adhesive properties. In the present study, the binding of isolated F4ab, F4ac and F4ad fimbriae, and F4ab/ac/ad-fimbriated E. coli, to glycosphingolipids from erythrocytes and from porcine small intestinal epithelium was examined, in order to get a comprehensive view of the F4-binding glycosphingolipids involved in F4-mediated hemagglutination and adhesion to the epithelial cells of porcine intestine. Specific interactions between the F4ab, F4ac and F4ad fimbriae and both acid and non-acid glycosphingolipids were obtained, and after isolation of binding-active glycosphingolipids and characterization by mass spectrometry and proton NMR, distinct carbohydrate binding patterns were defined for each fimbrial subtype. Two novel glycosphingolipids were isolated from chicken erythrocytes, and characterized as GalNAcα3GalNAcß3Galß4Glcß1Cer and GalNAcα3GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer. These two compounds, and lactosylceramide (Galß4Glcß1Cer) with phytosphingosine and hydroxy fatty acid, were recognized by all three variants of F4 fimbriae. No binding of the F4ad fimbriae or F4ad-fimbriated E. coli to the porcine intestinal glycosphingolipids occurred. However, for F4ab and F4ac two distinct binding patterns were observed. The F4ac fimbriae and the F4ac-expressing E. coli selectively bound to galactosylceramide (Galß1Cer) with sphingosine and hydroxy 24:0 fatty acid, while the porcine intestinal glycosphingolipids recognized by F4ab fimbriae and the F4ab-fimbriated bacteria were characterized as galactosylceramide, sulfatide (SO(3)-3Galß1Cer), sulf-lactosylceramide (SO(3)-3Galß4Glcß1Cer), and globotriaosylceramide (Galα4Galß4Glcß1Cer) with phytosphingosine and hydroxy 24:0 fatty acid. Finally, the F4ad fimbriae and the F4ad-fimbriated E. coli, but not the F4ab or F4ac subtypes, bound to reference gangliotriaosylceramide (GalNAcß4Galß4Glcß1Cer), gangliotetraosylceramide (Galß3GalNAcß4Galß4Glcß1Cer), isoglobotriaosylceramide (Galα3Galß4Glcß1Cer), and neolactotetraosylceramide (Galß4GlcNAcß3Galß4Glcß1Cer).

High prevalence of F4+ and F18+ Escherichia coli in Cuban piggeries as determined by serological survey.

Little information is available on the prevalence of swine enteropathogens in Cuba where diarrheic diseases are responsible for 31% and 37% of the total mortality during the neonatal and postweaning periods. F4+ and F18+ enterotoxigenic Escherichia coli and F18+ verotoxigenic E. coli induce diarrhea and edematous disease in pigs, but their distribution has never been thoroughly studied in the Cuban swine population. Therefore, the present study estimated the prevalence of F4- and F18-specific antibodies in sera of 1,044 6-month-old gilts distributed in 34 piggeries spread over the Cuban territory. For the data analysis, which included the optical density of individual samples tested by ELISA, random-effects models and a mixture model in R (package "mixAK"; Komárek, Computational Statistics and Data Analysis 53:3932-3947, 2009) were fitted. Low, moderate, and high levels of F4-specific antibodies were found in 67.6%, 26.8%, and 5.6% of the gilts, while 66.4% and 33.6% of gilts showed low and high levels of F18-specific antibodies. Hereby, we show that F4+ and F18+ E. coli are highly prevalent as potential enteropathogens in Cuban piggeries.

Recognition of blood group ABH type 1 determinants by the FedF adhesin of F18-fimbriated Escherichia coli.

F18-fimbriated Escherichia coli are associated with porcine postweaning diarrhea and edema disease. Adhesion of F18-fimbriated bacteria to the small intestine of susceptible pigs is mediated by the minor fimbrial subunit FedF. However, the target cell receptor for FedF has remained unidentified. Here we report that F18-fimbriated E. coli selectively interact with glycosphingolipids having blood group ABH determinants on type 1 core, and blood group A type 4 heptaglycosylceramide. The minimal binding epitope was identified as the blood group H type 1 determinant (Fucalpha2Galbeta3GlcNAc), while an optimal binding epitope was created by addition of the terminal alpha3-linked galactose or N-acetylgalactosamine of the blood group B type 1 determinant (Galalpha3(Fucalpha2)Galbeta3GlcNAc) and the blood group A type 1 determinant (GalNAcalpha3(Fucalpha2)-Galbeta3GlcNAc). To assess the role of glycosphingolipid recognition by F18-fimbriated E. coli in target tissue adherence, F18-binding glycosphingolipids were isolated from the small intestinal epithelium of blood group O and A pigs and characterized by mass spectrometry and proton NMR. The only glycosphingolipid with F18-binding activity of the blood group O pig was an H type 1 pentaglycosylceramide (Fucalpha2Galbeta3GlcNAc-beta3Galbeta4Glcbeta1Cer). In contrast, the blood group A pig had a number of F18-binding glycosphingolipids, characterized as A type 1 hexaglycosylceramide (GalNAcalpha3(Fucalpha2)Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer), A type 4 heptaglycosylceramide (GalNAcalpha3(Fucalpha2)Galbeta3GalNAcbeta3Galalpha4Galbeta4Glcbeta1Cer), A type 1 octaglycosylceramide (GalNAcalpha3(Fucalpha2)Galbeta3GlcNAcbeta3Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer), and repetitive A type 1 nonaglycosylceramide (GalNAcalpha3(Fucalpha2)Galbeta3GalNAcalpha3-(Fucalpha2)Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer). No blood group antigen-carrying glycosphingolipids were recognized by a mutant E. coli strain with deletion of the FedF adhesin, demonstrating that FedF is the structural element mediating binding of F18-fimbriated bacteria to blood group ABH determinants.

The possibility of positive selection for both F18(+)Escherichia coli and stress resistant pigs opens new perspectives for pig breeding.

F18(+)Escherichia coli infections causing post-weaning diarrhoea and/or oedema disease are a major cause of economic losses in pig industry. To date, no preventive strategy can protect pigs from F18(+)E. coli infections. One of the most attractive approaches to eliminate F18(+)E. coli infections is the selection for pigs that are resistant to F18(+)E. coli infections. However, this strategy was not believed to be favourable because of reports of genetic association with the stress-susceptibility gene in the Swiss Landrace. To investigate this potential association more thoroughly, 131 randomly selected Belgian hybrid pigs were genotyped for both the F18(+)E. coli resistance alleles (FUT1(A)) and the stress-susceptibility alleles (RYR1(T)) and their association was investigated by determining the linkage disequilibrium. This linkage disequilibrium (LD=-0.0149) is close to zero and does not differ significantly from 0 (likelihood ratio test chi(1)(2)=1.123, P=0.29), demonstrating no association between the FUT1(A) and RYR1(T) alleles. Furthermore, only a small fraction (4.6%) of the Belgian pigs was found to be resistant to F18(+)E. coli infections. Our results suggest that selection for F18(+)E. coli resistant pigs might be an attractive approach to prevent pigs from F18(+)E. coli infections, unlike to what has previously been postulated.