Rob H. Meloen

Immumohistochemical Detection and Localization of Prion Protein in Brain Tissue of Sheep With Natural Scrapie

A converted form of the normal cellular prion protein (PrP) accumulates in the brains of sheep with scrapie. We describe an immunohistochemical method for identifying scrapie-associated PrP (PrPSc) in periodate-lysine-paraformaldehyde-fixed brain tissue, which provides adequate preservation of tissue morphology. After pretreatment of tissue sections with formic acid and hydrated autoclaving, we located PrPSc in the brains of 50 sheep with natural scrapie by use of antipeptide antisera raised against ovine PrP. No PrP was seen in 20 sheep without histopathologic signs of scrapie. PrP80 that did not stain for amyloid was present in the cytoplasm and at the cell membrane of both neurons and astrocytes. Large amounts of PrPSc were seen at the cell membrane of neurons in the medulla oblongata and pons, whereas PrPSc accumulated at the cell membrane of astrocytes of the glial limitans in all brain regions. PrPSc that stained for amyloid was located in the walls of blood vessels and perivascularly in the brains of 32 (64%) of 50 sheep, mainly in the thalamus and never in the pons or medulla oblongata. No apparent topographic relationship existed between PrPSc that stained for amyloid and PrPSc accumulation associated with neurons or astrocytes. In all scrapie-affected sheep, PrPSc was present in brain regions with vacuolation, but it could also be detected in regions with minimal or no vacuolation. We conclude that the immunohistochemical detection of PrP can be an important confirmative test in scrapie diagnosis.

Rapid and Quantitative Cyclization of Multiple Peptide Loops onto Synthetic Scaffolds for Structural Mimicry of Protein Surfaces

Structure-based design of synthetic peptide-based molecules that mimic the functional site of natural proteins, plays an important role in drug discovery nowadays. Their application is widespread, ranging from synthetic antiviral, 6] antifertility, 2, 7] or antitumor 7, 8] agents to therapeutic agents that are able to mimic or disrupt 10] protein–protein interactions. A variety of structural mimics exist for a-helices, 12] b-turns or hairpins, 13] and b-sheets. 14] However, more complex topologies, like four-helix bundles, are often needed in order to mimic protein function adequately. The total synthesis of such complex structures is generally demanding; this limits their application and emphasizes the need for high-efficiency synthetic strategies. In this communication, we describe a onestep procedure for the immobilization of (multiple) peptide loops onto a synthetic scaffold (Scheme 1) starting from a linear peptide. The reaction is extremely fast and clean and runs very well with linear peptides that are 2–30 amino acids long (>30 not tested). It is compatible with all possible unprotected side-chain functionalities (except for free cysteine). It therefore avoids the need for complex synthetic strategies and this makes the reaction highly versatile with a very wide scope. As part of our research program on the mapping and reconstruction of the discontinuous epitope of follicle-stimulating hormone (FSH), which is a heterodimeric member of the cysteine-knot protein family, we recently discovered the fast and quantitative cyclization of dicysteine-containing peptides upon their treatment with a,a’-dibromoxylenes (T2). In organic solvents such as ACN, the reaction is rather slow and unselective, but it becomes unusually fast and entirely selective for cysteines when performed in aqueous solutions. For example, treatment of a 0.5 mm solution of the peptide *CRVPGDAHHADSLC# (1 a, where * = acetyl and # = amide) with 1.05 equiv of m-T2 in a 1:7 mixture of ACN/NH4HCO3 (20 mm, pH 7.8) gives the corresponding monocyclic product 2 a with >80 % yield in less than 15 min at RT (see Table 1). The corresponding intramolecular SS-dimer 3 is not formed (<5 %) as oxidative cyclization is not competitive under these conditions. There is no doubt that the reaction takes place exclusively at the free sulfhydryl groups, since corresponding peptides without sulfhydryl groups do not react at all with T2 scaffolds in the solvent system used. The difference in reactivity amongst various dicysteine-containing peptides that we have studied is negligible. The half-lives of peptides 1 a–g in the reaction with m-T2 vary only slightly (t1/2 = 1.4– 3.0 min, see Table 1), despite the fact that their length (14–42) and the number of amino acids that separate the two cysteines (0–22) are very different. In sharp contrast to this, there is a large difference in reactivity amongst different scaffolds. o-T2 (average t1/2 = 1.4 min) is slightly more reactive than mT2 (average t1/2 = 2.2 min), but both react much faster than p-T2 (average t1/2 = 8.6 min). Even with a fivefold excess of m-T2 the reaction predominantly gives the monocyclic product 2, whereas the 1:2 product 3 (Scheme 2) is formed with <10 % yield. Cyclization of dicysteine-containing peptides is a two-steps process: initial formation of linear intermediate 4 (Scheme 2) followed by intramolecular cyclization to give cyclic peptide 2. All intermediates (o-4, m-4, and p-4) are highly reactive and cyclize rapidly at low concentration, but their reactivity does not follow that of the template itself. For example, intermediates o-4 and p-4 are the most reactive by far and can only be observed by HPLC for larger ring sizes (e.g. , peptide 1 f), whereas m-4 is significantly more stable and was observed in significant amounts (= 15 %) for most peptides (see Table 1). Most likely, intermediates o-4 and p-4 are activated by a stabilizing resonance-effect in which the sulfur of the first thioether bond activates the second bromomethyl group for nucleophilic attack. Scheme 1. Schematic representation of the one-step synthesis of single-, double-, and tripleloop peptide constructs by treating di-, tri-, and tetracysteine containing peptides with bis-,

Structural aspects of antibody-antigen interaction revealed through small random peptide libraries

SummaryTwo small random peptide libraries, one composed of 4550 dodecapeptides and one of 8000 tripeptides, were synthesized in newly developed credit-card format miniPEPSCAN cards (miniPEPSCAN libraries). Each peptide was synthesized in a discrete well (455 peptides/card). The two miniPEPSCAN libraries were screened with three different monoclonal antibodies (Mabs). Two other random peptide libraries, expressed on the wall of bacteria (recombinant libraries) and composed of 107 hexa- and octapeptides, were screened with the same three Mabs. The aim of this study was to compare the amino acid sequence of peptides selected from small and large pools of random peptides and, in this way, investigate the potential of small random peptide libraries. The screening of the two miniPEPSCAN libraries resulted in the identification of a surprisingly large number of antibody-binding peptides, while the screening of the large recombinant libraries, using the same Mabs, resulted in the identification of only a small number of peptides. The large number of peptides derived from the small random peptide libraries allowed the determination of consensus sequences. These consensus sequences could be related to small linear and nonlinear parts of the respective epitopes. The small number of peptides derived from the large random peptide libraries could only be related to linear epitopes that were previously mapped using small libraries of overlapping peptides covering the antigenic protein. Thus, with respect to the cost and speed of identifying peptides that resemble linear and nonlinear parts of epitopes, small diversity libraries based on synthetic peptides appear to be superior to large diversity libraries based on expression systems.

Inactivated recombinant plant virus protects dogs from a lethal challenge with canine parvovirus.

A vaccine based upon a recombinant plant virus (CPMV-PARVO1), displaying a peptide derived from the VP2 capsid protein of canine parvovirus (CPV), has previously been described. To date, studies with the vaccine have utilized viable plant chimaeric particles (CVPs). In this study, CPMV-PARVO1 was inactivated by UV treatment to remove the possibility of replication of the recombinant plant virus in a plant host after manufacture of the vaccine. We show that the inactivated CVP is able to protect dogs from a lethal challenge with CPV following parenteral immunization with the vaccine. Dogs immunized with the inactivated CPMV-PARVO1 in adjuvant displayed no clinical signs of disease and shedding of CPV in faeces was limited following CPV challenge. All immunized dogs elicited high titres of peptide-specific antibody, which neutralized CPV in vitro. Levels of protection, virus shedding and VP2-specific antibody were comparable to those seen in dogs immunized with the same VP2- peptide coupled to keyhole limpet hemocyanin (KLH). Since plant virus-derived vaccines have the potential for cost-effective manufacture and are not known to replicate in mammalian cells, they represent a viable alternative to current replicating vaccine vectors for development of both human and veterinary vaccines.

Effective induction of neutralizing antibodies with the amino terminus of VP2 of canine parvovirus as a synthetic peptide

Fourteen synthetic peptides corresponding to previously mapped antigenic sites in VP2 of canine parvovirus (CPV) were used for immunization of rabbits to identify antiviral properties favourable for inclusion into a vaccine. Most antipeptide antisera obtained were reactive with viral protein, and with one of them it was possible to locate the hypothetical amino terminus of VP3 within positions 15-31 of VP2. Virus-neutralizing antibodies were only obtained with two overlapping 15-mer peptides corresponding in sequence to the amino terminus of VP2 (MSDGAVQPDGGQPAVRNERAT). Antibodies in the neutralizing sera bound most strongly to amino acids of the sequence DGGQPAV within the N-terminus of VP2, indicating that efforts to develop a synthetic vaccine against CVP should be focused on this stretch of amino acids. The two peptides induced long-lasting immunity (at least 8 months) using either Freunds adjuvant or aluminium hydroxide plus Quil A. Thus, this approach delineated the exact peptide sequence useful for vaccines applied to the amino-terminal region of VP2. These findings in experimental animals form a solid basis for exploration of a synthetic peptide vaccine against parvovirus infection in dogs, minks or cats.

Full protection in mink against mink enteritis virus with new generation canine parvovirus vaccines based on synthetic peptide or recombinant protein

Two recently developed vaccine--one based on synthetic peptide and one based on recombinant capsid protein--fully protected dogs against heavy experimental canine parvovirus (CPV) infection. The high sequence homology ( > 98%) and antigenic similarity between CPV and mink enteritis virus (MEV), feline panleukopenia virus, and raccoon parvovirus, suggest that both vaccines could protect mink, cats and raccoons against these respective host range variants. This was tested in mink and turned out to be the case. The two vaccines were fully protective and as effective as a conventional commercial vaccine based on inactivated virus. Surprisingly, this protection was obtained after only a single injection. Furthermore, the vaccinal dose of 150 micrograms of conjugated peptide or 3 micrograms of recombinant VP2 particles per animal, are sufficiently low to be cost-effective and applicable on a large scale.

Synthetic Peptide Vaccines: Success at Last

Peptide vaccines would form the ideal ultimate vaccine because they are safe both in production as well as application, easy to handle, store and transport. Peptide vaccines have the advantage that they can be produced in a completely reproducible manner, they are cheap compared to subunit or whole protein vaccines and potentially they can be precisely targeted to meet specific demands. For instance, peptide vaccines may be used to break maternal immunity or raise very specific antibodies for research or diagnostics. Furthermore synthetic peptide vaccines offer the potential possibility for multivalent vaccines and to incorporate adjuvanticity. Notwithsanding numerous attempts only one peptide vaccine has found its way to the market. This peptide vaccine is however not targeted to a pathogen but to a self-protein, Gonadotropin Releasing Hormone (GnRH), and used to immunocastrate production, animals (Hoskinson et al, 1990). In this contribution we shall discuss why peptide vaccines for pathogens, notably viruses, have failed so far, by focusing on Foot-and-Mouth Disease Virus (FMDV). For FMDV substantial attempts have been made to develop a synthetic peptide vaccine. These attempts have failed. However an attempt to develop a peptide vaccine against CPV was successful and may help to learn how failures with other attempts can be overcome.

Performance and hormone levels of immunocastrated, surgically castrated and intact male pigs fed ad libitum high- and low-energy diets

Immunocastration by immunisation against gonadotrophin-releasing hormone (GnRH), is a good alternative for surgical castration in male pigs, both to prevent boar taint and for animal welfare reasons. In this study we investigated the effect of immunocastration on growth performance of pigs from 25 to 110 kg fed two diets differing in NEf content. Sixty piglets were divided into 20 blocks. Each block consisted of three littermates: a boar, a surgical castrate and an immunocastrate. Two blocks were housed in each pen. The pigs were fed ad libitum and individual feed intake was recorded with an automatic feed registration device. A high-energy and a low-energy diet were fed (9.70 and 8.30 MJ NEf/kg, respectively, based on tabulated values). The immunocastrates were immunised at 10 and 17 weeks of age with 62.5 μg d-Lys6-GnRH tandem dimer peptide conjugated to ovalbumin in Specol adjuvant. Plasma testosterone levels in immunocastrates decreased after the first injection and reached non-detectable levels at 20 weeks of age and thereafter. Immunocastrates had non-detectable fat androstenone levels at slaughter. The results of the concomitant digestibility experiments showed that the estimated NEf contents of the diets were 9.64 and 9.04 MJ/kg, respectively. No differences were observed in digestibility of proximate nutrients among sexes, but digestibility of Ca and P was higher for boars and immunocastrates as compared to surgical castrates (P<0.05). There was no interaction between type of diet and sex for growth performance characteristics except for energy conversion ratio, that approached significance (P=0.095). Feed intake for immunocastrates and surgical castrates was similar, and was higher than for boars on both diets (P<0.001). There was a tendency towards a better growth rate for immunocastrates compared to surgical castrates, irrespective of the energy level of the diet. When compared to boars, growth rate was higher in both immunocastrates and surgical castrates, which was significant only for the low-energy diet (P<0.01). Energy conversion ratio (ECR; expressed as MJ NEf/kg gain) was better for boars than for surgical castrates (P<0.05). For immunocastrates, ECR was in between that of boars and surgical castrates, and was better than for surgical castrates on the low-energy diet (P<0.05). Meat percentage was higher for boars than for surgical castrates and immunocastrates (P<0.05). On the high-energy diet, meat percentage was lower in immunocastrates than in surgical castrates (P<0.05) but on the low-energy diet it was similar. The results of the present study demonstrate that there was a tendency towards a better growth performance for immunocastrates as compared to surgical castrates, especially on the low-energy diet.

Active immunization against gonadotrophin-releasing hormone in Chinese male pigs: effects of dose on antibody titer, hormone levels and sexual development

The objective of this study was to determine the optimal dose of a GnRH vaccine for immunocastration of Chinese male pigs, based on immune, endocrine and testicular responses. Forty-two crossbred (Chinese Yanan x Large White) male pigs were randomly assigned to one of the five treatments as follows: (I) 0 microg (control, n=8); (II) 10 microg (n=8); (III) 62.5 microg (n=8); (IV) 125 microg (n=8); (V) 250 microg (n=10), D-Lys6-GnRH tandem dimer (TDK) peptide equivalent of conjugate (TDK-OVA), using Specol as the adjuvant. Pigs were immunized at 13 and 21 weeks of age and were slaughtered at 31 weeks of age. Blood samples for antibody titer and hormone assays were collected at 13, 21, 24 and 31 weeks of age. At these time-points, testis size was also measured. At slaughter, testis weight was recorded and fat samples were collected for androstenone assay. Four animals, one out of each immunized group, responded poorly to the immunization (non-responders). At slaughter, serum testosterone and LH levels, fat androstenone levels and testis size/weight of these non-responders were similar to those in control animals. Antibody titers of non-responders were substantially lower (P<0.05) than in other immunized pigs. For the animals that responded well to the immunization (immunocastrated pigs), serum testosterone and LH levels, fat androstenone levels and testis size or weight were reduced (P<0.05) as compared to either controls or non-responders, at all doses tested. There was a significant effect of dose of TDK-OVA on antibody titers. The overall mean antibody titers in the 62.5 or 125 microg dose group (53.6 and 50.5% binding, respectively) were significantly higher than in the 10 or 250 microg group (39.2 and 40.24% binding, respectively). At slaughter, there was a significant dose effect on testis size or weight and on serum testosterone levels, but there was no dose effect on serum LH levels and fat androstenone levels. Testis size or weight in the 10 microg group was reduced to a lesser extent (P<0.05) than in the three higher dose groups. At slaughter, in comparison to controls, mean testis size of immunocastrated pigs in treatments II-V was reduced to 55, 21, 33 and 25%, respectively, whereas testis weight was reduced to 39, 12, 18 and 14%, respectively. Reduction of testis size and/or weight is important for visual assessment of castration at the slaughterline, therefore, it is concluded that a dose of 10 microg peptide is not suitable. We conclude that, within the dose-range studied, the 62.5 microg dose is optimal for future GnRH immunization studies or future practical use in immunocastration of Chinese male pigs.

Effects of active immunization against GnRH on serum LH, inhibin A, sexual development and growth rate in Chinese female pigs

Surgical castration of young female pigs is common practice in Chinese pig farming today. The purpose of the present study is to investigate anti-GnRH immunization as a practical alternative to surgical castration for female pigs. Thirty-six Chinese female crossbred pigs (Chinese Yanan x Yorkshire) were selected from 12 litters, three pigs from each litter, at the age of 10-13 weeks. One pig from each litter was immunized with 62.5 microg D-Lys6-GnRH-tandem-dimer peptide conjugated to ovalbumin in Specol adjuvant at Week 0 (0 week post-vaccination, wpv), and a booster vaccination was given 8 weeks later (8 wpv). Its intact and castrate littermates (surgically castrated at the time of weaning, i.e. at 6 weeks of age) were administered the vehicle and served as controls. Antibody titers, serum LH and inhibin A were determined at the day of first vaccination, every 4 weeks thereafter and at the day of slaughter (18 wpv). At slaughter, ovaries were inspected for the presence of follicles and corpora lutea, and ovarian and uterine weights were recorded. Ten of twelve immunized pigs responded well to the immunization (immunocastrated animals), while the remaining two pigs responded poorly (nonresponders). Antibody titres in immunocastrated animals steadily increased after immunization, became maximal at 12 wpv and remained high until slaughter. Serum LH levels were reduced (P < 0.05) in immunocastrated pigs as compared to intact controls and surgical castrates. Serum inhibin A levels decreased after vaccination, and equaled surgical castrate levels from 8 wpv until the end of the experiment. Ovarian and uterine weights (1.3 +/- 0.2 and 43.9 +/- 11.4 g, respectively; mean +/- S.E.M.) were significantly lower (P < 0.05) in immunocastrates than in intact controls (9.4 +/- 1.1 and 390.9 +/- 67.2 g, respectively). Antibody titers were significantly lower (P < 0.05) in nonresponders than in immunocastrated pigs from 12 wpv to slaughter. Ovarian and uterine weights were similar in nonresponders and in intact controls. Macroscopically, no follicular structures were found in ovaries of immunocastrated pigs, while large follicles or corpora lutea were observed in the ovaries of both nonresponders and intact controls. Although not significant, immunocastrates had a numerically higher average daily gain than surgical castrates and intact controls (0.74 +/- 0.04 versus 0.66 +/- 0.04 versus 0.66 +/- 0.03 kg per day, respectively; mean +/- S.E.M., P = 0.09). Results obtained in the present study demonstrate that anti-GnRH immunization can be an attractive alternative to surgical castration for Chinese crossbred female pigs. Our results also question the beneficial effect of surgical castration on growth as compared to intact controls.