H.P.D. Fijten

Highly pathogenic or low pathogenic avian influenza virus subtype H7N1 infection in chicken lungs: small differences in general acute responses

Avian influenza virus can be divided into two groups, highly pathogenic avian influenza virus (HPAI) and low pathogenic avian influenza virus (LPAI) based on their difference in virulence. To investigate if the difference in clinical outcome between LPAI and HPAI in chickens is due to immunological host responses in the lung within the first 24 hours post infection (hpi), chickens were infected with LPAI or HPAI of subtype H7N1. Virus was found in the caudal and cranial part of the lung. With LPAI, virus was localised around the intrapulmonary bronchus and secondary bronchi. In sharp contrast, HPAI was detected throughout the whole lung. However, based on viral RNA levels, no quantitative difference was observed between LPAI and HPAI infected birds. In infected areas of the lungs, an influx of CD8α+ cells as well as KUL01+ macrophages and dendritic cells (DC) occurred as fast as 8 hpi in both infected groups. No major difference between LPAI and HPAI infected birds in the induction of cytokines and interferons at mRNA level in lung tissue was found.In conclusion, the differences in lethality for chickens infected with LPAI or HPAI could be ascribed to difference in location of the virus. However similar amounts of viral RNA, similar cytokine mRNA levels, and similar influxes of CD8α+ and KUL01+ macrophages and DC were found between HPAI and LPAI in the lungs. A cytokine storm at mRNA level as described for mammals was not observed in the lungs of HPAI infected birds within 24 hpi.

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 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.

Lung pathogenicity of European genotype 3 strain porcine reproductive and respiratory syndrome virus (PRRSV) differs from that of subtype 1 strains.

Abstract Porcine reproductive and respiratory syndrome (PRRS) is difficult to control due to a high mutation rate of the PRRS virus (PRRSV) and the emergence of virulent strains. The objective of this study was to analyse early and late pathological responses in the respiratory tract after infection with the European PRRSV subtype 3 strain Lena in comparison to two European PRRSV subtype 1 strains: Belgium A and Lelystad-Ter Huurne (LV). For each virus strain, groups of twelve pigs were inoculated, and four pigs per group were euthanized at days 3, 7 and 35 post-infection (p.i.) for consecutive examination. Infection with strain Lena resulted in a more severe disease than with the subtype 1 strains, an inflammatory response within the first week of infection with expression of IL-1α in the lung and lymph node, and an influx of neutrophils and monocytes in bronchoalveolar lavage fluid (BALF). Infection with strain Belgium A or LV resulted in mild or no pathology within the first week of infection, but inflammatory cell influx in the lung interstititium was increased at the end of the experiment at day 35 p.i. At five weeks p.i., all strains induced a higher percentage of cytotoxic T cells and higher levels of IFN-γ producing cells in BALF. This might have contributed to clearance of virus. In general, subtype 3 strain Lena induced a stronger early inflammatory response which led to more severe clinical disease and pathology. On the other hand, this may have supported an enhanced or faster clearance of virus in tissues, compared to subtype 1 strains.

Phenotypic modulation and cytokine profiles of antigen presenting cells by European subtype 1 and 3 porcine reproductive and respiratory syndrome virus strains in vitro and in vivo.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes continuous problems in the pig industry, due to high costs of outbreaks and reduced welfare of diseased pigs. The severity of infection is, partly, dependent on the virus strain. Recently isolated Eastern-European subtype 3 strains are more pathogenic than the widespread subtype 1 strains. There is, however, almost no information available about the mechanisms involved in the pathogenicity of these subtype 3 strains. The objective of the present study was to characterize the in vitro and in vivo response of two European subtype 1 strains, Belgium A and Lelystad-Ter Huurne (LV), and a virulent subtype 3 strain, Lena, in bone marrow-derived dendritic cells (BM-DC) (in vitro) and alveolar macrophages (in vitro and in vivo). It was shown that infection with the Lena strain resulted in a higher apoptosis of cells in vitro and a higher level of infectivity in vitro and in vivo than the other virus strains. Furthermore, infection with Lena resulted in a small downregulation of the immunologically relevant cell surface molecules SLA-I, SLA-II and CD80/86 in vitro, and SLA-II in vivo. In spite of these differences, in vitro cytokine responses did not differ significantly between strains, except for the absence of IL-10 production by Lena in BM-DC. The higher infectivity, apoptosis and downregulation of the cell surface molecules, may have contributed to the increased pathogenicity of Lena, and have dampened specific immune responses. This could explain the delayed and decreased adaptive immune responses observed after infections with this strain.

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.

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.

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.

Towards a peptide-based suspension array for the detection of pestivirus antibodies in swine

Classical swine fever (CSF) is a highly contagious and lethal disease in swine. Serological tests for the diagnosis of CSF need not only to detect antibodies against CSFV, but also need to differentiate these from antibodies against other pestiviruses. To investigate the possibilities of specific peptide-based serology, various synthetic peptides that represent a well-described linear epitope of the CSFV E2 protein (TAVSPTTLR) were used to test the viability of a peptide-based suspension array for the detection of antibodies against pestiviruses in swine. The results show that N-terminally biotinylated peptides can bind to avidin conjugated beads, and function in detection of the corresponding monoclonal antibody WH303. There are indications that the length of the spacer between epitope and biotin affect the efficiency of the peptide-antibody interaction. A protocol was established that enables probing for antibodies in porcine sera, where neutravidin-blocking of serum and the use of empty control beads for normalization was crucial. With a set of porcine sera with antibodies against various pestiviruses, the proof of concept of a peptide-based suspension array for specific detection of antibodies against pestiviruses in porcine sera was demonstrated.

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.