Stabilizing effects of excipients on dissociation of intact (146S) foot-and-mouth disease virions into 12S particles during storage as oil-emulsion vaccine.

Most conventional foot-and-mouth disease virus (FMDV) vaccines contain oil-adjuvant. Their potency decreases upon prolonged storage. Intact (146S) FMDV particles can dissociate into 12S degradation products with a concomitant decrease in immunogenicity. We therefore measured virion stability in vaccines using two previously developed ELISAs to separately quantify 12S and 146S particles. Virions completely dissociated into 12S particles within 3 months after oil-emulsification. Dissociation occurred at a much lower rate in a comparable aqueous solution that was not oil-emulsified. Thus, oil-emulsification stimulates virion dissociation, presumably due to the protein denaturing effect of the oil-water interface. In real-time stability studies the stability of oil-adjuvanted virions of four different FMDV strains was significantly increased by addition of sucrose and BSA in a synergistic manner. Contrary to BSA addition, the effect of sucrose addition was concentration dependent. This study illustrates the importance of analysing antigen integrity after oil-emulsification and provides methods for FMDV vaccine stabilization.

Effect of natural and chimeric haemagglutinin genes on influenza A virus replication in baby hamster kidney cells.

Baby hamster kidney (BHK21) cells are used to produce vaccines against various viral veterinary diseases, including rabies and foot-and-mouth-disease. Although particular influenza virus strains replicate efficiently in BHK21 cells the general use of these cells for influenza vaccine production is prohibited by the poor replication of most strains, including model strain A/PR/8/34 [H1N1] (PR8). We now show that in contrast to PR8, the related strain A/WSN/33 [H1N1] (WSN) replicates efficiently in BHK21 cells. This difference is determined by the haemagglutinin (HA) protein since reciprocal reassortant viruses with swapped HAs behave similarly with respect to growth on BHK21 cells as the parental virus from which their HA gene is derived. The ability or inability of six other influenza virus strains to grow on BHK21 cells appears to be similarly dependent on the nature of the HA gene since reassortant PR8 viruses containing the HA of these strains grow to similar titres as the parental virus from which the HA gene was derived. However, the growth to low titres of a seventh influenza strain was not due to the nature of the HA gene since a reassortant PR8 virus containing this HA grew efficiently on BHK21 cells. Taken together, these results suggest that the HA gene often primarily determines influenza replication efficiency on BHK21 cells but that in some strains other genes are also involved. High virus titres could be obtained with reassortant PR8 strains that contained a chimeric HA consisting of the HA1 domain of PR8 and the HA2 domain of WSN. HA1 contains most antigenic sites and is therefore important for vaccine efficacy. This method of producing the HA1 domain as fusion to a heterologous HA2 domain could possibly also be used for the production of HA1 domains of other viruses to enable the use of BHK21 cells as a generic platform for veterinary influenza vaccine production.

Mutations in the M-gene segment can substantially increase replication efficiency of NS1 deletion influenza A virus in MDCK cells.

Influenza viruses unable to express NS1 protein (delNS1) replicate poorly and induce large amounts of interferon (IFN). They are therefore considered candidate viruses for live-attenuated influenza vaccines. Their attenuated replication is generally assumed to result from the inability to counter the antiviral host response, as delNS1 viruses replicate efficiently in Vero cells, which lack IFN expression. In this study, delNS1 virus was parallel passaged on IFN-competent MDCK cells, which resulted in two strains that were able to replicate to high virus titers in MDCK cells due to adaptive mutations especially in the M-gene segment but also in the NP and NS gene segments. Most notable were clustered U-to-C mutations in the M segment of both strains and clustered A-to-G mutations in the NS segment of one strain, which presumably resulted from host cell-mediated RNA editing. The M segment mutations in both strains changed the ratio of M1 to M2 expression, probably by affecting splicing efficiency. In one virus, 2 amino acid substitutions in M1 additionally enhanced virus replication, possibly through changes in the M1 distribution between the nucleus and the cytoplasm. Both adapted viruses induced levels of IFN equal to that of the original delNS1 virus. These results show that the increased replication of the adapted viruses is not primarily due to altered IFN induction but rather is related to changes in M1 expression or localization. The mutations identified in this paper may be used to enhance delNS1 virus replication for vaccine production.

MDCK cell line with inducible allele B NS1 expression propagates delNS1 influenza virus to high titres.

Influenza A viruses lacking the gene encoding the non-structural NS1 protein (delNS1) have potential use as live attenuated vaccines. However, due to the lack of NS1, virus replication in cell culture is considerably reduced, prohibiting commercial vaccine production. We therefore established two stable MDCK cell lines that show inducible expression of the allele B NS1 protein. Upon induction, both cell lines expressed NS1 to about 1000-fold lower levels than influenza virus-infected cells. Nevertheless, expression of NS1 increased delNS1 virus titres to levels comparable to those obtained with an isogenic virus strain containing an intact NS1 gene. Recombinant NS1 expression increased the infectious virus titres 244 to 544-fold and inhibited virus induced apoptosis. However, NS1 expression resulted in only slightly, statistically not significant, reduced levels of interferon-ß production. Thus, the low amount of recombinant NS1 is sufficient to restore delNS1 virus replication in MDCK cells, but it remains unclear whether this occurs in an interferon dependent manner. In contrast to previous findings, recombinant NS1 expression did not induce apoptosis, nor did it affect cell growth. These cell lines thus show potential to improve the yield of delNS1 virus for vaccine production.

Effect of thiomersal on dissociation of intact (146S) foot-and-mouth disease virions into 12S particles as assessed by novel ELISAs specific for either 146S or 12S particles.

Intact (146S) foot-and-mouth disease virions (FMDVs) can dissociate into specific (12S) viral capsid degradation products. Using two single-domain antibody fragments that bind specifically to either 146S or 12S particles we developed two ELISAs for the quantification of these particles in FMDV antigen preparations used for vaccine manufacturing. Only O serotype strains are detected in the 146S specific ELISA whereas strains of most serotypes are detected in the 12S specific ELISA. However, the 146S concentration of A and Asia 1 serotype strains could be measured indirectly using the 12S specific ELISA by prior conversion of 146S into 12S particles by heat treatment. This allowed us to demonstrate that addition of the preservative thiomersal to FMDV antigens stimulates the dissociation into 12S particles of O, A and Asia 1 serotype strains upon prolonged storage at 4°C. FMDV dissociation is known to result in a strongly reduced immunogenicity, which was experimentally confirmed here. Therefore, we recommend to omit thiomersal from FMD vaccines to increase its shelf life.

Adaptation of a Madin-Darby canine kidney cell line to suspension growth in serum-free media and comparison of its ability to produce avian influenza virus to Vero and BHK21 cell lines.

Madin-Darby canine kidney (MDCK) cells are currently considered for influenza vaccine manufacturing. A drawback of these cells is their anchorage dependent growth, which greatly complicates process scale-up. In this paper a novel MDCK cell line (MDCK-SFS) is described that grows efficiently in suspension and retained high expression levels of both α-2,6 and α-2,3 sialic acid receptors, which bind preferably to human and avian influenza viruses, respectively. The production of avian influenza virus by BHK21, Vero and MDCK-SFS cell lines was compared. Although BHK21 cells consisted of two populations, one of which lacks the α-2,3 receptor, they supported the replication of two influenza strains to high titres. However, BHK21 cells are generally not applicable for influenza production since they supported the replication of six further strains poorly. MDCK-SFS cells yielded the highest infectious virus titres and virus genome equivalent concentration for five of the eight influenza strains analyzed and the highest hemagglutination activity for all eight virus strains. Taken together with their suitability for suspension growth this makes the MDCK-SFS cell line potentially useful for large scale influenza virus production.

Characterization of foot-and-mouth disease virus antigen by surface-enhanced laser desorption ionization-time of flight-mass spectrometry in aqueous and oil-emulsion formulations.

We have used a novel method, surface-enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS), to characterize foot-and-mouth disease virus (FMDV) vaccine antigens. Using specific capture with FMDV binding recombinant antibody fragments and tryptic digestion of FMDV antigens the spectral peaks representing the FMDV structural proteins VP1, VP2, VP3 and VP4 were identified. VP1 existed as 2 variants differing by 0.2kDa and VP4 as 8 variants differing by 14-17Da. Such heterogeneities have not been reported earlier. They could represent oxidation of VP4 and N-glycation of VP1. We also detected FMDV proteolysis upon incubation at elevated temperatures and impurities in FMDV antigen preparations. Finally, we could also characterize FMDV antigen present in emulsions with oil adjuvant by SELDI-TOF-MS. Such FMDV antigen retained the VP4 protein which is known to be specifically present in intact (146S) FMDV particles but absent from specific (12S) degradation products. This indicates that virions do not dissociate upon emulsification.

Enhancement of toxin- and virus-neutralizing capacity of single-domain antibody fragments by N-glycosylation.

Single-domain antibody fragments (VHHs) have several beneficial properties as compared to conventional antibody fragments. However, their small size complicates their toxin- and virus-neutralizing capacity. We isolated 27 VHHs binding Escherichia coli heat-labile toxin and expressed these in Saccharomyces cerevisiae. The most potent neutralizing VHH (LT109) was N-glycosylated, resulting in a large increase in molecular mass. This suggests that N-glycosylation of LT109 improves its neutralizing capacity. Indeed, deglycosylation of LT109 decreased its neutralizing capacity three- to fivefold. We also studied the effect of glycosylation of two previously isolated VHHs on their ability to neutralize foot-and-mouth disease virus. For this purpose, these VHHs that lacked potential N-glycosylation sites were genetically fused to another VHH that was known to be glycosylated. The resulting fusion proteins were also N-glycosylated. They neutralized the virus at at least fourfold-lower VHH concentrations as compared to the single, non-glycosylated VHHs and at at least 50-fold-lower VHH concentrations as compared to their deglycosylated counterparts. Thus, we have shown that N-glycosylation of VHHs contributes to toxin- and virus-neutralizing capacity.

Passive immunization with llama single-domain antibody fragments reduces foot-and-mouth disease transmission between pigs.

We aim to develop a method that confers rapid protection against foot-and-mouth disease (FMD) by passive immunization with recombinant llama single-domain antibody fragments (VHHs). Previously constructed genetic fusions of two VHHs (VHH2s) that either neutralizes FMDV or binds to porcine immunoglobulin to increase the serum half-life, conferred only limited protection to pigs. We therefore now generated VHH3s containing an additional FMDV binding VHH. Two VHH3s neutralized FMDV more potently than single VHHs and were highly produced by yeast cells. Injection of a mixture of these two VHH3s 24h before FMD challenge infection of pigs reduced and delayed the development of clinical disease, viraemia and viral shedding. Furthermore, it significantly (P=0.023) delayed FMD transmission. Thus, we have shown a proof of concept of passive FMD immunoprophylaxis using VHHs.

Passive immunization of pigs with bispecific llama single-domain antibody fragments against foot-and-mouth disease and porcine immunoglobulin.

Foot-and-mouth disease (FMD) is a contagious viral disease of cloven-hoofed animals that occasionally causes outbreaks in Europe. We aim to develop an immunotherapy that confers rapid protection against FMD in outbreak situations. For this purpose, we previously isolated llama single-domain antibody fragments (VHHs) binding to FMDV or porcine immunoglobulin (pIg). The pIg binding VHHs can be genetically fused to other VHHs, resulting in so-called VHH2s. As compared to non-pIg binding VHHs such VHH2s have a 100-fold increased serum half-life which is essential for effective immunotherapy. We have now produced three bispecific VHH2s by fusion of three FMDV binding VHHs (clones M3, M8 and M23) to a pIg binding VHH (VI-4). The resulting yeast-produced VHH2s bound FMDV and pIg with high affinity (K(D) about 1nM) and neutralized FMDV in vitro as efficiently as their monovalent counterparts. To evaluate their therapeutic potential all three VHH2s were intramuscularly injected into pigs that were challenge infected with FMDV 24h later. Administration of one of these VHH2s (M23ggsVI-4) reduced the viremia significantly (P=0.0034) and reduced viral shedding almost significantly (P=0.11). However, it did not prevent development of clinical signs or transmission of FMDV. These results suggest that immunotherapy using bispecific VHH2s binding to FMDV and pIg is possible in principle, but should be improved by increasing VHH2 dosage or using more potent VHH2s.

Properties, production, and applications of camelid single-domain antibody fragments.

Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies) have several advantages for biotechnological applications. They are well expressed in microorganisms and have a high stability and solubility. Furthermore, they are well suited for construction of larger molecules and selection systems such as phage, yeast, or ribosome display. This minireview offers an overview of (1) their properties as compared to conventional antibodies, (2) their production in microorganisms, with a focus on yeasts, and (3) their therapeutic applications.

Passive immunization of guinea pigs with llama single-domain antibody fragments against foot-and-mouth disease.

Foot-and-mouth disease (FMD) is a highly contagious disease that occasionally causes outbreaks in Europe. There is a need for therapies that provide rapid protection against FMD in outbreak situations. We aim to provide such rapid protection by passive immunization with llama single-domain antibody fragments (VHHs). Twenty-four VHHs binding serotype O FMDV in vitro were isolated from immunized llamas by phage display and expressed in bakers yeast for further characterization. They recognized four functionally independent antigenic sites. Six strongly FMDV neutralizing VHHs bound to a peptide representing the GH-loop of viral protein 1 known to be involved in binding to the cellular receptor of FMDV. Clone M8, recognizing this antigenic site, and clone M23, recognizing another antigenic site, showed synergistic in vitro virus neutralization. Three FMDV specific VHHs were PEGylated in order to decrease their rapid blood clearance and thus enable in vivo guinea pig protection experiments. Passive immunization with individual VHHs showed no protection, but a mixture of M8 and M23 showed partial transient protection. The protection afforded by these VHHs was however low as compared to the complete protection afforded by convalescent guinea pig serum. In contrast, these VHHs showed far more efficient in vitro FMDV neutralization than convalescent guinea pig serum. This lack of correlation between in vitro neutralization and in vivo protection lends further credence to the notion that opsonophagocytosis of FMDV is important for protection in vivo.

[A case of very serious Sarcoptes mange in alpacas (Lama pacos)]. / Een geval van ernstige Sarcoptes-schurft bij alpaca's (Lama pacos).

After the diagnosis sarcoptic mange in four alpaca's (Lama pacos) we have tried to control this infection. Despite three treatments with doramectin, three with ivermectin, four with amitraz and two with diazinon we were unable to get the animals free of Sarcoptes mites and their condition deteriorated. One animal died six month after the first treatment. The three remaining animals were euthanized one month thereafter.

Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy.

We previously demonstrated that oral application of the recombinant single-domain antibody fragment (VHH) clone K609, directed against Escherichia coli F4 fimbriae, reduced E. coli-induced diarrhoea in piglets, but only at high VHH doses. We have now shown that a large portion of the orally applied K609 VHH is proteolytically degraded in the stomach. Stringent selection for proteolytic stability identified seven VHHs with 7- to 138-fold increased stability after in vitro incubation in gastric fluid. By DNA shuffling we obtained four clones with a further 1.5- to 3-fold increased in vitro stability. These VHHs differed by at most ten amino acid residues from each other and K609 that were scattered over the VHH sequence and did not overlap with predicted protease cleavage sites. The most stable clone, K922, retained 41% activity after incubation in gastric fluid and 90% in jejunal fluid. Oral application of K922 to piglets confirmed its improved proteolytic stability. In addition, K922 bound to F4 fimbriae with higher affinity and inhibited fimbrial adhesion at lower VHH concentrations. K922 is thus a promising candidate for prevention of piglet diarrhoea. Furthermore, our findings could guide selection and improvement by genetic engineering of other recombinant antibody fragments for oral use.

Escherichia coli F4 fimbriae specific llama single-domain antibody fragments effectively inhibit bacterial adhesion in vitro but poorly protect against diarrhoea.

Oral administration of polyclonal antibodies directed against enterotoxigenic Escherichia coli (ETEC) F4 fimbriae is used to protect against piglet post-weaning diarrhoea. For cost reasons, we aim to replace these polyclonal antibodies by recombinant llama single-domain antibody fragments (VHHs) that can be produced efficiently in microorganisms. Six F4 fimbriae specific VHHs were isolated. The VHH that was produced at the highest level by yeast, K609, was further analysed. 3.8 mg/L K609 inhibited 90% of bacterial attachment to intestinal brush borders in vitro. Perfusion of a jejunal segment with at least 4 mg/L K609 reduced the ETEC-induced fluid loss, but only to 30%. Preventive administration of a high K609 dose (150 mg/(piglet day)) to piglets that were challenge infected with ETEC resulted in less severe diarrhoea only at 4 and 5 days post-infection, but did not improve average daily weight gain, ETEC shedding and piglet survival. Thus, we have shown that an antibody fragment that effectively inhibited in vitro ETEC adhesion to intestinal brush borders poorly protected piglets against experimental ETEC infection.

Stimulation of chymosin secretion by simultaneous expression with chymosin-binding llama single-domain antibody fragments in yeast.

We studied the effect of coexpression of chymosin and chymosin-binding llama single-domain antibody fragments (VHHs) on the secretion of chymosin by Saccharomyces cerevisiae cells. A VHH expression library containing chymosin-specific VHHs was obtained by immunization of a llama and coexpressed with chymosin in yeast. From this library, we obtained two VHH clones that stimulated chymosin secretion by screening colonies for the level of chymosin secreted. These VHHs bound biotinylated chymosin in an immunoblot procedure but failed to bind chymosin in ELISA, suggesting that their interaction with chymosin was of low affinity. In a second approach, chymosin-specific VHHs were first selected using phage display and then coexpressed with chymosin in yeast cells. Screening yeast cells for higher levels of chymosin secretion resulted in 11 VHHs. Sequence analysis revealed that these 11 VHHs formed four sets of related VHHs that were different from the previously isolated two VHHs. Although binding of VHHs to chymosin could not be demonstrated in ELISA using soluble VHHs, it could be unambiguously demonstrated for clones isolated by phage display, using phage-displayed VHHs. Finally, quantitative Western blot analysis of chymosin amounts demonstrated that coexpression with VHH domains can stimulate the level of secreted chymosin 1.5- to 6-fold.

Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease.

A diagnostic ELISA with recombinant Fasciola hepatica cathepsin L-like protease as antigen was developed to detect antibodies against F. hepatica in sheep and cattle. The recombinant cathepsin L-like protease was generated by functional expression of the cDNA from adult stage F. hepatica flukes in Saccharomyces cerevisiae. Specificity and sensitivity of the cathepsin L enzyme-linked immunosorbent assay (ELISA) was assessed using sera from sheep and calves experimentally or naturally mono-infected with F. hepatica and six-seven other parasites. The sensitivity of the cathepsin L ELISA for sheep and cattle sera was 99.1 and 100%, respectively. In the experimental setting with established mono-infections, the specificity of the cathepsin L ELISA was 98.5% for cattle sera and 96.5% for sheep sera. In experimentally infected cattle and sheep, the first detection of F. hepatica-specific antibodies appeared first between 5 and 7 weeks post-infection, but depended on the infectious dose of F. hepatica. In ELISA the detection preceded first detection of the infection based on egg counts and remained detectable till at least 23 weeks after a primary F. hepatica infection. Detection of Fasciola gigantica infections was similar to detection of F. hepatica. The first detection occurred at week 5 and signals persisted for at least 20 weeks. All sera from naturally F. hepatica infected sheep were seropositive in the cathepsin L-like ELISA. The relevance of this ELISA format was also evaluated using sera from naturally infected cattle in the Netherlands, Ecuador and Vietnam and compared with results from egg-counts. For the latter two endemic areas with mixed parasitic infections the 'apparent' sensitivity of the cathepsin L ELISA was calculated for all serum samples together to be 90.2%. The 'apparent' specificity under these conditions was calculated to be 75.3%. In cattle, the cathepsin L ELISA was superior to the concurrently evaluated peptide ELISA format using a single epitope as the antigen both in controlled natural infections as well as in infections in endemic areas. The present ELISA-format contributes a relatively sensitive and reliable tool for the early serodiagnosis of bovine and ovine fasciolosis.

Llama heavy-chain V regions consist of at least four distinct subfamilies revealing novel sequence features.

In addition to conventional antibodies (Abs), camelids possess Abs consisting of only heavy chains. The variable domain of such a heavy-chain Ab (VHH) is fully capable of antigen (Ag) binding. Earlier analysis of 47 VHHs showed sequence features unique to VHH domains. These include the presence of characteristic amino acid substitutions in positions which, in conventional VH domains are involved in interdomain interactions, and the presence of a long third complementarity-determining region (CDR3) which is frequently constrained by an interloop disulphide bond. Here, we describe a large (152) set of Lama glama VHH cDNAs. Based on amino acid sequence similarity, these and other published camelid VHHs were classified into four subfamilies. Three subfamilies are absent in dromedaries, which have been the primary source of VHHs thus far. Comparison of these subfamilies to conventional VH regions reveals new features characteristic of VHHs and shows that many features earlier regarded as characteristic of VHHs in general are actually subfamily specific. A long CDR3 with a concomitant putative additional disulphide bond is only observed in two VHH subfamilies. Furthermore, we identified new VHH-characteristic residues at positions forming interdomain sites in conventional VH domains. The VHH subfamilies also differ from each other and conventional VH domains in the canonical structure of CDR1 and CDR2, mean CDR3 length, and amino acid residue variability. Since different VHH-characteristic residues are observed in all four subfamilies, these subfamilies must have evolved independently from classical VH domains.

Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies.

Antigen specific llama VHH antibody fragments were compared to antigen specific mouse monoclonal antibodies with respect to specificity, affinity and stability. The llama VHH antibody fragments and the mouse monoclonal antibodies investigated were shown to be highly specific for the protein antigen hCG or the hapten antigen RR-6. The affinity of the interaction between monovalent llama VHH antibody fragments and their antigen is close to the nanomolar range, similar to the bivalent mouse monoclonal antibodies studied. Llama VHH antibody fragments are similar to mouse monoclonal antibodies with respect to antigen binding in the presence of ammonium thiocyanate and ethanol. The results show that relative to antigen specific mouse monoclonal antibodies, antigen specific llama VHH fragments are extremely temperature stable. Two out of six llama VHHs are able to bind antigen specifically at temperatures as high as 90 degrees C, whereas four out of four mouse monoclonal antibodies are not functional at this temperature. Together with the finding that llama VHH fragments can be produced at high yield in Saccharomyces cerevisiae, these findings indicate that in the near future antigen specific llama VHH fragments can be used in for antibodies unexpected products and processes.

Expression of the house dust mite allergen Der p 2 in the baker's yeast Saccharomyces cerevisiae.

BACKGROUND AND RESULTS: The major house dust mite allergen Der p 2 was expressed as a recombinant mature protein in the baker's yeast Saccharomyces cerevisiae. The yeast produces the protein fused to the invertase signal peptide, leading to the secretion of Der p 2 as a soluble protein into the culture medium. The signal peptide is hereby cleaved off, resulting in a mature allergen. In this system Der p 2 was produced in 7.6 (+/-2.9) mg/L growth culture. Purification of the recombinant allergen was achieved by a single gel filtration step, resulting in a purity > or = 95%. The yeast-derived Der p 2 was almost indistinguishable from natural Der p 2 with respect to IgE-reactivity and binding to the majority of Der p 2 specific MoAbs -- as was shown in RAST analysis (n = 168) and a sandwich ELISA and RIA analysis, respectively. Recombinant and natural Der p 2 also showed similar biological activity in histamine release assays (n = 4). CONCLUSION: An expression system for Der p 2 was developed that enables the production of a soluble allergen in the culture supernatant with immunological characteristics similar to the natural allergen. In addition, yeast offers the advantage of the absence of endotoxin in comparison to E. coli. This might facilitate acceptance of recombinant allergens for in vivo applications as immunotherapy or skin-prick testing.