Eberhard Pfaff

Synthetic peptide vaccines yield monoclonal antibodies to cellular and pathological prion proteins of ruminants.

Transmissible spongiform encephalopathies are closely linked to the accumulation of a pathological isoform of a host-encoded prion protein (PrP(C)), designated PrP(Sc). In an attempt to generate mono- and polyclonal antibodies to ruminant PrP, 32 mice were vaccinated with peptide vaccines which were synthesized according to the amino acid sequence of ovine PrP. By this approach five PrP-reactive polyclonal antisera directed against four different domains of the protein were stimulated. Splenocytes of mice which had developed PrP-reactive antibodies were used for the generation of monoclonal antibodies (MAbs). Obtained PrP-specific MAbs were directed to three different domains of ruminant PrP which differed from the three previously described major MAb binding sites in rodent PrP. MAbs exhibited reactivity with non-denatured ruminant PrP(C) in ELISA and immunoprecipitation and with denatured ovine and bovine PrP(Sc) in immunoblot. Cross-reactivity was observed with PrP(C) of nine other mammalian species and with pathological PrP preferably of ruminants and weakly with that of hamster and mouse. The generated MAbs will be useful tools for the development of diagnostic tests for BSE and scrapie as well as for pathogenesis studies of these diseases.

Establishment of persistent hepatitis C virus infection and replication in vitro.

Hepatitis C virus (HCV) is a major cause of chronic viral hepatitis. Development of anti-viral strategies has been hampered by the lack of efficient cell systems to propagate HCV in vitro. To establish a long-term culture system, we tested human hepatoma (HuH7, HepG2) and porcine non-hepatoma (PK15, STE) cell lines, as well as several culture and infection conditions. As a marker for virus replication, minus-strand HCV RNA in infected cells was detected by an enhanced detection system using nested RT-PCR followed by hybridization analysis. Short-term efficiency of HCV infection (10 days) was slightly increased by addition of polyethylene glycol (PEG) and/or dimethyl sulfoxide (DMSO) to culture media during inoculation of HuH7, PK15 and STE cells, but no augmentation in long-term culture was achieved, suggesting enhanced attachment of HCV to cells rather than more efficient infection. A stabilizing effect on HCV propagation was observed for 50 days in a serum-free medium with stimulation of the low-density lipoprotein (LDL) receptor expression by lovastatin. Using partially serum-free culture conditions, long-term persistence of HCV in cells and release of virions into supernatant was achieved for up to 130 days. Infectivity of released virions in supernatants after long-term culturing (day 30-80) was shown by successful infection of fresh cells. In conclusion, supplementation with PEG, DMSO and lovastatin during inoculation did not enhance virus replication substantially, but continued stimulation of LDL-receptor expression resulted in infections which persisted for over 4 months. These data support the hypothesis of an LDL-receptor mediated uptake of HCV into cells in vitro.

Multiple Amino Acid Residues within the Rabbit Prion Protein Inhibit Formation of Its Abnormal Isoform

ABSTRACT Transmissible spongiform encephalopathies (TSEs) are neurological diseases that are associated with the conversion of the normal host-encoded prion protein (PrP-sen) to an abnormal protease-resistant form, PrP-res. Transmission of the TSE agent from one species to another is usually inefficient and accompanied by a prolonged incubation time. Species barriers to infection by the TSE agent are of particular importance given the apparent transmission of bovine spongiform encephalopathy to humans. Among the few animal species that appear to be resistant to infection by the TSE agent are rabbits. They survive challenge with the human kuru and Creutzfeldt-Jakob agents as well as with scrapie agent isolated from sheep or mice. Species barriers to the TSE agent are strongly influenced by the PrP amino acid sequence of both the donor and recipient animals. Here we show that rabbit PrP-sen does not form PrP-res in murine tissue culture cells persistently infected with the mouse-adapted scrapie agent. Unlike other TSE species barriers that have been studied, critical amino acid residues that inhibit PrP-res formation are located throughout the rabbit PrP sequence. Our results suggest that the resistance of rabbits to infection by the TSE agent is due to multiple rabbit PrP-specific amino acid residues that result in a PrP structure that is unable to refold to the abnormal isoform associated with disease.

Identification of T-cell epitopes in the structural and non-structural proteins of classical swine fever virus.

To identify new T-cell epitopes of classical swine fever virus (CSFV), 573 overlapping, synthetic pentadecapeptides spanning 82% of the CSFV (strain Glentorf) genome sequence were synthesized and screened. In proliferation assays, 26 peptides distributed throughout the CSFV viral protein sequences were able to induce specific T-cell responses in PBMCs from a CSFV-Glentorf-infected d/d haplotype pig. Of these 26 peptides, 18 were also recognized by PBMCs from a CSFV-Alfort/187-infected d/d haplotype pig. In further experiments, it could be shown that peptide 290 (KHKVRNEVMVHWFDD), which corresponds to amino acid residues 1446-1460 of the CSFV non-structural protein NS2-3 could induce interferon-gamma secretion after secondary in vitro restimulation. The major histocompatibility complex (MHC) restriction for stimulation of T-cells by this pentadecapeptide was identified as being mainly MHC class II and partially MHC class I. In cytolytic assays, CSFV-specific cytotoxic T-lymphocytes (CTLs) were able to lyse peptide 290-loaded target cells. These findings indicate the existence of a CSFV-specific helper T-cell epitope and a CTL epitope in this peptide.

Identification of Foot-and-Mouth Disease Virus-Specific Linear B-Cell Epitopes To Differentiate between Infected and Vaccinated Cattle

ABSTRACT Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals. For several years, vaccination of animals, which had proven to be successful for the eradication of the disease, has been forbidden in the United States and the European Community because of the difficulty of differentiating between vaccinated and infected animals. In this study, detailed investigations of the bovine humoral immune response against FMD virus (FMDV) were performed with the aim of identifying viral epitopes recognized specifically by sera derived from FMDV-infected animals. The use of overlapping 15-mer synthetic peptides, covering the whole open reading frame of FMDV strain O1K in a peptide enzyme-linked immunosorbent assay, allowed the identification of 12 FMDV strain O1K-specific linear B-cell epitopes. Six of these linear B-cell epitopes, located in the nonstructural proteins, were used in further assays to compare the reactivities of sera from vaccinated and infected cattle. Antibodies recognizing these peptides could be detected only in sera derived from infected cattle. In further experiments, the reactivity of the six peptides with sera from animals infected with different strains of FMDV was tested, and strain-independent infection-specific epitopes were identified. Thus, these results clearly demonstrate the ability of a simple peptide-based assay to discriminate between infected and conventionally FMD-vaccinated animals.

Characterization of porcine T lymphocytes and their immune response against viral antigens

T lymphocytes play a central role in the antigen-specific immune response against various pathogens. To detect and to characterize porcine T lymphocytes, monoclonal antibodies (mAb) against leukocyte differentiation antigens had been raised and classified for their specificity. Analyses of porcine T lymphocytes with specific mAb against CD4 and CD8 differentiation antigens revealed differences in the composition of the porcine T-lymphocyte population compared to other species. In addition to the known subpopulations, CD4+CD8- T helper cells and CD4-CD8+ cytolytic T lymphocytes, extra-thymic CD4+CD8+ T lymphocytes and a substantial proportion of CD2-CD4-CD8- T cell receptor (TcR)-gamma delta+ T cells could be detected in swine. Functional analyses of porcine T-lymphocyte subpopulations revealed the existence of two T-helper cell fractions with the phenotype CD4+CD8- and CD4+CD8+. Both were reactive in primary immune responses in vitro, whereas only cells derived from the CD4+CD8+ T-helper-cell subpopulation were able to respond to recall antigen in a secondary immune response. With regard to T lymphocytes with cytolytic activities, two subsets within the CD4-CD8+ T-cell subpopulation could be defined by the expression of CD6 differentiation antigens: CD6- cells which showed spontaneous cytolytic activity and CD6+ MHC I-restricted cytolytic T lymphocytes including virus-specific cytolytic T lymphocytes. These results enable now a detailed view into the porcine T-cell population and the reactivity of specific T cells involved in the porcine immune response against pathogens. Furthermore this knowledge offers the possibility to investigate specific interactions of porcine T lymphocytes with virus-specific epitopes during vaccination and viral infections.

Antigenic features of prion proteins of sheep and of other mammalian species.

Pathological prion protein (PrPSc) which is a conformational isoform of a host-encoded protein designated (PrPC) serves as a specific marker protein for the immunochemical diagnosis of transmissible spongiform encephalopathies (TSE). The generation of suitable antibodies to PrPSc therefore underlies the specificity and sensitivity of diagnostic assays. However, most antibodies reported to date are directed to a limited number of epitopes only. PrPC is a highly conserved cell membrane protein in all mammalian species studied to date. In an attempt to generate antibodies to further regions of PrP we raised antisera in rabbits and chicken against sixteen synthetic peptides which represent the complete aminoacid sequence of ovine PrP. By this approach immunotolerance was overcome and immunoblot-reactive antibodies were stimulated to epitopes at almost any site of ovine PrPC and PrPSc. A large number of different antibodies cross-reacted also with affinity-purified PrPCs from other mammalian species including cow, goat, pig, man, dog, cat, mink, mouse, hamster and guinea pig. No epitope, however, was recognized exclusively on the pathological or cellular isoform of PrP indicating that both isoforms occur in highly denatured conformations on the immunoblots. Antibodies to the amino-terminus are suitable for immunoprecipitation of PrP. The availability of rabbit and chicken anti-peptide antibodies to PrP will greatly improve immunochemical diagnosis and pathogenetic studies on these diseases.

Dual Nature of the Infectious Prion Protein Revealed by High Pressure

Crude brain homogenates of terminally diseased hamsters infected with the 263K strain of scrapie (PrPSc) and purified prion fibrils were heated or pressurized at 800 megapascals and 60 °C for 2 h in different buffers and in water. Prion proteins (PrP) were analyzed for their proteinase K resistance in immunoblots and for their infectivity in hamster bioassays. A notable decrease in the proteinase K resistance of unpurified prion proteins, probably because of pressure-induced changes in the protein conformation of native PrPSc or the N-truncated PrP-(27–30), could be demonstrated when pressurized at initially neutral conditions in several buffers and in water but not in a slightly acidic pH. A subsequent 6–7 log10 reduction of infectious units/g in phosphate-buffered saline buffer, pH 7.4, was found. The proteinase K-resistant core was also not detectable after purification of prions extracted from pressurized samples, confirming pressure effects at the level of the secondary structure of prion proteins. However, opposite results were found after pressurizing purified prions, arguing for the existence of pressure-sensitive β-structures (PrPScΔPsen) and extremely pressure-resistant β-structures (PrPScΔPres). Remarkably, after the first centrifugation step at 540,000 × g during isolation, prions remained proteinase K-resistant when pressurized in all tested buffers and in water. It is known that purified fibrils retain infectivity, but the isolated protein (full and N-truncated) behaved differently from native PrPSc under pressure, suggesting a kind of semicrystalline polymer structure.

Studies on a species-specific epitope in murine, ovine and bovine prion protein.

Transmissible spongiform encephalopathies are fatal neurodegenerative disorders which are linked to abnormal isoforms of the prion protein (PrP), which is expressed in different cells of various mammalian species. Susceptibility to disease and reduced transmission rates upon the first passage to another species are thought to be a result of functional and biochemical differences of the PrP as a consequence of amino acid sequence among species. In 1985 an epidemic of bovine spongiform encephalopathy (BSE) started after accidental transmission of scrapie by feeding infected sheep and goat meat and bone meal products to cattle. In this report we present data demonstrating species-specific epitopes in bovine, ovine and murine PrP that are based on amino acid substitutions at positions 108 and 110. Rabbit antisera to synthetic peptides representing amino acid sequence 108 to 123 of PrP of cattle, sheep and mice reacted strongly with modified PrP of the homologous host but not, or only poorly, with PrP of heterogeneous origin. Cross-reactivity was observed, however, with antisera to bovine and ovine peptide sequences 102 to 117, thus stressing the importance of the location of the amino acid substitution in synthetic peptides used for immunization. Based on these data, BSE PrP and ovine and murine scrapie PrP can be distinguished from each other, and these differences might help elucidate the species barrier effect.

The Porcine Humoral Immune Response against Pseudorabies Virus Specifically Targets Attachment Sites on Glycoprotein gC

ABSTRACT High titers of virus-neutralizing antibodies directed against glycoprotein gC of Pseudorabies virus (PRV) (Suid herpesvirus 1) are generally observed in the serum of immunized pigs. A known function of the glycoprotein gC is to mediate attachment of PRV to target cells through distinct viral heparin-binding domains (HBDs). Therefore, it was suggested that the virus-neutralizing activity of anti-PRV sera is directed against HBDs on gC. To address this issue, sera with high virus-neutralizing activity against gC were used to characterize the anti-gC response. Epitope mapping demonstrated that amino acids of HBDs are part of an antigenic antibody binding domain which is located in the N-terminal part of gC. Binding of antibodies to this antigenic domain of gC was further shown to interfere with the viral attachment. Therefore, these results show that the viral HBDs are accessible targets for the humoral anti-PRV response even after tolerance induction against self-proteins, which utilize similar HBDs to promote host protein-protein interactions. The findings indicate that the hosts immune system can specifically block the attachment function of PRV gC. Since HBDs promote the attachment of a number of herpesviruses, the design of future antiherpesvirus vaccines should aim to induce a humoral immune response that prevents HBD-mediated viral attachment.