GnRH tandem peptides for inducing an immunogenic response to GnRH-I without cross-reactivity to other GnRH isoforms.

Gonadotropin releasing hormone (GnRH) occurs in various isoforms in mammals, i.e. GnRH-I (mammalian GnRH), GnRH-II (chicken GnRH-II), GnRH-III (salmon GnRH) and two forms of lamprey GnRH. The function of the latter four molecules have only been partially investigated. Also not much is known about the physiological effects of GnRH-I immunization on the function of these GnRH isoforms. In order to avoid possible harmful side-effects due to undesired neutralization of GnRH isoforms, GnRH-I specificity of antibodies raised against a panel of alternative GnRH antigens was determined. The results show that GnRH antigens can be designed which generate antibodies that specifically bind GnRH-I, without cross-reacting with other GnRH isoforms.

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.

A structural model of pestivirus E(rns) based on disulfide bond connectivity and homology modeling reveals an extremely rare vicinal disulfide.

E(rns) is a pestivirus envelope glycoprotein and is the only known viral surface protein with RNase activity. E(rns) is a disulfide-linked homodimer of 100 kDa; it is found on the surface of pestivirus-infected cells and is secreted into the medium. In this study, the disulfide arrangement of the nine cysteines present in the mature dimer was established by analysis of the proteolytically cleaved protein. Fragments were obtained after digestion with multiple proteolytic enzymes and subsequently analyzed by liquid chromatography-electrospray ionization mass spectrometry. The analysis demonstrates which cysteine is involved in dimerization and reveals an extremely rare vicinal disulfide bridge of unknown function. With the assistance of the disulfide arrangement, a three-dimensional model was built by homology modeling based on the alignment with members of the Rh/T2/S RNase family. Compared to these other RNase family members, E(rns) shows an N-terminal truncation, a large insertion of a cystine-rich region, and a C-terminal extension responsible for membrane translocation. The homology to mammalian RNase 6 supports a possible role of E(rns) in B-cell depletion.

The role of the individual amino acids of a GnRH-tandem-dimer peptide used as an antigen for immunocastration of male piglets determined with systematic alanine replacements.

Immunocastration targeting gonadotropin releasing hormone (GnRH) can be obtained in male piglets using native GnRH conjugates. However, due to insufficient efficacy of these conjugates, improved GnRH antigens, like peptides existing of repeats of the GnRH amino acid sequence, have been designed. We previously reported about a dimerised GnRH-tandem peptide with a D-Lys at position 6 of the native GnRH sequence (G6k-TD) being highly effective. To evaluate the contribution of each individual amino acid of the GnRH decapeptide to the efficacy of the G6k-TD peptide, each amino acid was replaced consecutively by alanine (Ala-scan). The G6k-TD peptides were conjugated to ovalbumin, used for immunisation and tested for their ability to elicit GnRH antibodies and to immunocastrate male piglets. The results show that four out of nine amino acids (pGlu-1, Ser-4, Arg-8 and Gly-10) can be replaced by alanine without negatively affecting immunocastration efficacy. Replacement of amino acids in other positions (Tyr-5, Leu-7 and Pro-9) gave partial decrease of efficacy, respectively, five, six and six out of seven piglets were immunocastrated. Replacements at two other positions (His-2 and Trp-3) completely negated immunocastration activity. Thus, seven out of nine amino acid positions in the basic unit of G6k-TD can be substituted by alanine without affecting immunocastration efficacy.

The nature of the bond between peptide and carrier molecule determines the immunogenicity of the construct.

The influence of the nature of the bond between a peptide and a (lipidic) carrier molecule on the immunogenicity of that construct was investigated. As types of bonds a thioester-, a disulfide-, an amide- and a thioether bond were investigated. As carrier molecules a peptide, an N-palmitoylated peptide or a C(16)-hydrocarbon chain were used. The biostability of the bond between peptide and carrier molecule is thioether > amide > disulfide >> thioester. However, the immunogenic potency of the constructs used was found to be thioester > disulfide > amide > thioether. In conclusion, a construct with a bond between peptide and (lipidic) carrier molecule that is more susceptible to biological degradation is more immunogenic when used in a peptide-based vaccine than a bond that is less susceptible to biological degradation.

Active immunization against gonadotrophin-releasing hormone in Chinese male pigs.

We have investigated, under the normal conditions of local Chinese pig farming, castration of young male pigs by vaccination with a newly developed vaccine against gonadotrophin releasing hormone (GnRH). Because of the very early onset of puberty, long fattening period and relatively harsh circumstances in Chinese pig production, an investigation of the endocrine response of Chinese breeds to this type of vaccination was of particular interest. Fifteen crossbred boars (Yorkshire x Yanan) from three different litters were randomly assigned to three groups of five animals each. The first group was immunized at 13 weeks of age with a GnRH tandem dimer OVA-conjugate in Specol and received a booster immunization 8 weeks later. The second group was injected with Specol alone and served as untreated controls. The remaining group was surgically castrated at the time of weaning (at 6 weeks of age). Pigs were fed ad libitum from weaning onwards. All animals were slaughtered at 31 weeks of age. Immunized boars had undetectable or low serum testosterone (0.09 +/- 0.12 ng/ml), low fat androstenone (0.05 +/- 0.01 microg/g) levels and very low testes weights (19.1 +/- 4.3 g). Intact controls had much higher serum levels of testosterone (9.76 +/- 4.81 ng/ml), fat androstenone levels (2.26 +/- 0.87 microg/g) and testes weights (114.3 +/- 29.41 g) at slaughter. Both the immunized and castrated group grew significantly faster than intact boars (p < 0.01). Average daily gains in immunized, castrated and intact animals were 0.69 +/- 0.08, 0.63 +/- 0.05 and 0.42 +/- 0.07 kg (mean +/- SD), respectively. The present data demonstrate for the first time that the newly developed anti-GnRH vaccine works very well under practical Chinese pig farming conditions, and can be an attractive alternative to surgical castration.

Synthetic peptides derived from the beta2-beta3 loop of Raphanus sativus antifungal protein 2 that mimic the active site.

Rs-AFPs are antifungal proteins, isolated from radish (Raphanus sativus) seed or leaves, which consist of 50 or 51 amino acids and belong to the plant defensin family of proteins. Four highly homologous Rs-AFPs have been isolated (Rs-AFP1-4). The structure of Rs-AFP1 consists of three beta-strands and an alpha-helix, and is stabilized by four cystine bridges. Small peptides deduced from the native sequence, still having biological activity, are not only important tools to study structure-function relationships, but may also constitute a commercially interesting target. In an earlier study, we showed that the antifungal activity of Rs-AFP2 is concentrated mainly in the beta2-beta3 loop. In this study, we synthesized linear 19-mer peptides, spanning the entire beta2-beta3 loop, that were found to be almost as potent as Rs-AFP2. Cysteines, highly conserved in the native protein, are essential for maintaining the secondary structure of the protein. Surprisingly, in the 19-mer loop peptides, cysteines can be replaced by alpha-aminobutyric acid, which even improves the antifungal potency of the peptides. Analogous cyclic 19-mer peptides, forced to adopt a hairpin structure by the introduction of one or two non-native disulfide bridges, were also found to possess high antifungal activity. The synthetic 19-mer peptides, like Rs-AFP2 itself, cause increased Ca2+ influx in pregerminated fungal hyphae.

Synthetic peptide vaccines: unexpected fulfillment of discarded hope?

In the early eighties it was realized that the ultimate vaccine would be a synthetic peptide. Major efforts were put into the development of a synthetic vaccine for foot-and-mouth disease virus (FMDV) for which even today no alternative exists besides the classical vaccine based on inactivated virus. Despite impressive progress, a peptide vaccine that could match the classical vaccine with respect to efficacy (i.e. full protection of all animals after a single vaccination) has not materialized. This has led to the belief that synthetic vaccines were not possible. However, in the early nineties we developed a synthetic peptide vaccine for canine parvovirus that did match the classical vaccine based on inactivated virus (i.e. protected all animals). Based on the difference of FMDV (an RNA virus) and canine parvovirus (a DNA virus), we suggested that in the case of FMDV, more than one antigenic site should be used, instead of the single one used previously. In our opinion multiple sites are necessary to prevent the development of escape mutants of FMDV. Unfortunately, the additional sites of FMDV are highly discontinuous. Until recently it was impossible to reconstruct these sites in the form of synthetic peptides. In the past few years, new methods have been developed that allow recombination of such sites into synthetic molecules. If successfully applied to FMDV, synthetic peptide vaccines and many others may become feasible in the near future. Moreover, the ability to mimic complex discontinuous sites by synthetic peptides will have a major impact on the rapidly developing area of therapeutic vaccines.

Synthetic peptides as functional mimics of a viral discontinuous antigenic site.

Functional reproduction of discontinuous antigenic site D of foot-and-mouth disease virus (FMDV) has been achieved by means of synthetic peptide constructions that integrate into a single molecule each of the three protein loops that define the antigenic site. The site D mimics are designed on the basis of the X-ray structure of FMDV type C-S8c1 with the aid of molecular dynamics, so that the five residues assumed to be involved in antigenic recognition are located on the same face of the molecule, exposed to solvent and defining a set of native-like distances and angles. The designed site D mimics are disulphide-linked heterodimers that consist of a larger unit containing VP2(71-84), followed by a polyproline module and by VP3(52-62), and a smaller unit corresponding to VP1(188-194). Guinea pig antisera to the peptides recognize the viral particle and compete with site D-specific monoclonal antibodies, while inoculation with a simple (non-covalently bound) admixture of the three VP1-VP3 sequences yields no detectable virus-specific serum conversion. Similar results have been reproduced in two cattle. Antisera to the peptides are also moderately neutralizing of FMDV in cell culture and partially protective of guinea pigs against challenge with the virus. These results demonstrate functional mimicry of the discontinuous site D by the peptides, which are therefore obvious candidates for a multicomponent peptide-based vaccine against FMDV.

Mucosal immunization with experimental feline immunodeficiency virus (FIV) vaccines induces both antibody and T cell responses but does not protect against rectal FIV challenge.

Feline immunodeficiency virus (FIV) is a natural lentiviral pathogen of cats which can be experimentally transmitted via rectal and vaginal routes--the major routes of human immunodeficiency virus type 1 transmission in man. An important objective for lentiviral research is the development of vaccine strategies which generate good mucosal immune responses capable of giving protection from a mucosal virus challenge. The experimental vaccines employed in this study were based on (a) a peptide from the third variable region of the FIV envelope glycoprotein and (b) fixed whole FIV, Glasgow-8 strain. Adjuvants used were Quil A and cholera toxin for mucosal administration and incomplete Freund's adjuvant and immune stimulating complexes for subcutaneous injection. Mucosal immunization was given by rectal and intranasal routes. Both antibody and proliferative responses were elicited by mucosal immunization and cholera toxin was found to be a good mucosal adjuvant. The addition of a lipo thioester to the FIV peptide improved IgG and IgA responses upon parenteral administration. However, no protection from a rectal FIV challenge was achieved.

Characterization of melanocortin receptor ligands on cloned brain melanocortin receptors and on grooming behavior in the rat.

Since the melanocortin MC3 and melanocortin MC4 receptors are the main melanocortin receptor subtypes expressed in rat brain, we characterized the activity and affinity of nine melanocortin receptor ligands using these receptors in vitro, as well as their activity in a well-defined melanocortin-induced behavior in the rat: grooming behavior. We report here that [D-Tyr4]melanotan-II and RMI-2001 (Ac-cyclo-[Cys4, Gly5, D-Phe7, Cys10]alpha-MSH-NH2) have significantly higher affinity and potency on the rat melanocortin MC4 receptor as compared to the rat melanocortin MC3 receptor. Nle-gamma-MSH (melanocyte-stimulating hormone) was the only ligand with higher affinity and potency on the rat melanocortin MC3 receptor. The potency order of melanocortin MC4 receptor agonists, but not that of melanocortin MC3 receptor agonists, fitted with the potency of these ligands to stimulate grooming behavior, when administered intracerebroventricularly. SHU9119 (Ac-cyclo-[Nle4, Asp5, D-Nal(2)7, Lys10]alpha-MSH-(4-10)-NH2) and RMI-2005 (Ac-cyclo-[Cys4, Gly5, D-Na](2)7, Nal(2)9, Cys10]alpha-MSH-(4-10)-NH2) were able to inhibit alpha-MSH-induced melanocortin receptor activity in vitro, as well as alpha-MSH-induced grooming behavior. Melanotan-II, [Nle4-D-Phe7]alpha-MSH and RMI-2001 were also effective in inducing grooming behavior when administered intravenously. In the absence of purely selective melanocortin MC(3/4) receptor ligands, we demonstrated that careful comparison of ligand potencies in vitro with ligand potencies in vivo, could identify which melanocortin receptor subtype mediated alpha-MSH-induced grooming behavior. Furthermore, blockade of novelty-induced grooming behavior by SHU9119 demonstrated that this physiological stress response is mediated via activation of the melanocortin system.

Conformation of the core sequence in melanocortin peptides directs selectivity for the melanocortin MC3 and MC4 receptors.

Melanocortin peptides regulate a variety of physiological processes. Five melanocortin receptors (MC-R) have been cloned and the MC3R and MC4R are the main brain MC receptors. The aim of this study was to identify structural requirements in both ligand and receptor that determine gamma-melanocyte-stimulating hormone (MSH) selectivity for the MC3R versus the MC4R. Substitution of Asp10 in [Nle4]Lys-gamma2-MSH for Gly10 from [Nle4]alpha-MSH, increased both activity and affinity for the MC4R while the MC3R remained unaffected. Analysis of chimeric MC3R/MC4Rs and mutant MC4Rs showed that Tyr268 of the MC4R mainly determined the low affinity for [Nle4]Lys-gamma2-MSH. The data demonstrate that Asp10 determines selectivity for the MC3R, however, not through direct side chain interactions, but probably by influencing how the melanocortin core sequence is presented to the receptor-binding pocket. This is supported by mutagenesis of Tyr268 to Ile in the MC4R which increased affinity and activity for [Nle4]Lys-gamma2-MSH, but decreased affinity for two peptides with constrained cyclic structure of the melanocortin core sequence, MT-II and [D-Tyr4]MT-II, that also displayed lower affinity for the MC3R. This study provides a general concept for peptide receptor selectivity, in which the major determinant for a selective receptor interaction is the conformational presentation of the core sequence in related peptides to the receptor-binding pocket.

Highly immunogenic and fully synthetic peptide-carrier constructs targetting GnRH.

To use peptides as synthetic vaccines, they have to be coupled to a carrier protein to make them more immunogenic. Coupling efficiency between a carrier protein and a peptide, however, is difficult to control with respect to loading density of the peptide. This makes these carrier proteins poorly suitable for practicle use. Attempts have been reported to find carrier molecules or delivery systems which allow easy coupling or incorporation of peptides, reproducible loading densities and well defined products. We compared several promising constructs or delivery systems by immunization of male pigs using a tandem GnRH peptide as a branched polylysine construct, a lipo-thioester, a lipo-amide or a KLH conjugate in CFA, and the lipoamide peptide in an immuno-stimulating complex (ISCOM). We found the lipo-thioester and the branched polylysine constructs to be the most effective carrier molecules for the induction of antibodies against GnRH and immunocastration of pigs.

In vitro inhibition of the bioactivity of follicle-stimulating hormone by antisera against a peptide representing part of the FSH-receptor.

The aim of the present work was to define an FSH receptor (FSHR) peptide that can induce antibodies that will inhibit the bioactivity of FSH. Therefore, the hFSHR sequence was aligned with that of all other known G-protein coupled receptors. An area with increased sequence homology was identified between the FSH-, LH-, TSH receptors, the C5a receptor and the IL8 receptor. The similarity consists of a richness in acidic (D and E) and hydrophobic (Y and F) residues. In hFSHR the sequence is EDNESSYSRGFDMTYTEFDYDLCNEVVD (amino acid 299-326). Research on both the C5a- and IL8-receptor has indicated that this part is responsible for hormone binding but not for signal transduction. Protamine. an antagonist for both the C5a- and IL8 receptor also inhibited the bioactivities of FSH and LH when tested in a bioassay. This suggests that in the hFSHR this region might also be involved in hormone binding. Specificity of this region towards the diverse ligands all binding to the C5a or to the IL8 receptor might be attributed to differences in the profile of alternating basic and hydrophobic residues. Therefore, the hypothesis was tested as to whether antisera raised against peptides of this FSHR-domain would inhibit FSH-bioactivity but not LH-bioactivity. Indeed antisera were found (anti-hFSHR 309-322) that inhibited the biological activity of FSH in a bioassay. These antisera proved to be specific since they did not inhibit the bioactivity of LH. These data suggest that the core sequence (hFSHR 309-322) of the aligned domain of the hFSHR, in analogy to the IL8- and C5a receptors, is involved in hormone binding and ligand specificity. This domain therefore forms a valuable tool in FSH- and FSHR research for scientific and medical purposes.

New GnRH-like peptide construct to optimize efficient immunocastration of male pigs by immunoneutralization of GnRH.

Castration of male pigs is routinely performed in order to prevent the occurrence of boar taint in pig carcasses. However, boar taint can also be eliminated by immunological castration using a synthetic peptide vaccine against GnRH. For pig farming, to make immunocastration a feasible alternative method to surgical castration, the composition of the vaccine has to be not only reliable and effective but also cost-efficient and safe. Previously the authors have developed an effective immunocastration vaccine by replacing the monomer GnRH by a much more immunogenic tandem peptide. However, this tandem-GnRH vaccine preparation needs Complete Freund's adjuvant and to be applied at a relatively high dose. Therefore, alternative antigens were designed to cope with this problem and tested with different adjuvants and dosages. An effective new antigen was designed based on a GnRH-tandem peptide, which was dimerized and modified in one amino acid position of the decapeptide to allow conjugation of this tandem-dimer to ovalbumin. In mild adjuvants and in low dosage, this antigen was very effective in reducing testis weight, serum LH and androstenone level in backfat. Thus, an improved immunocastration vaccine has been designed that is relatively cost-efficient and highly efficacious in two vaccinations at low dose.

Mapping the antigenic structure of porcine parvovirus at the level of peptides.

The antigenic structure of the capsid proteins of porcine parvovirus (PPV) was investigated. A total of nine linear epitopes were identified by Pepscan using porcine or rabbit anti-PPV antisera. No sites were identified with a panel of neutralising monoclonal antibodies (MAbs). All epitopes were located in the region corresponding to the major capsid protein VP2. Based on this information, and on analogy to other autonomous parvoviruses, 24 different peptides were synthesised, coupled to keyhole limpet haemocyanin (KLH) and used to immunise rabbits. Most antisera were able to bind viral protein. Only peptides from the N-terminal part of VP2 were able to induce virus-neutralising antibodies, although at low levels. A similar neutralising activity could be obtained in pigs. The exposure of the N-terminus was shown in full virions, both by immunoelectron microscopy and absorption experiments. It is concluded that in PPV, the VP2 N-terminus is involved in virus neutralisation (VN) and peptides from this region are therefore primary targets for developing peptide-based vaccines against this virus.

Synthetic peptide vaccines: palmitoylation of peptide antigens by a thioester bond increases immunogenicity.

Synthetic peptides have frequently been used to immunize animals. However, peptides less than about 20 to 30 amino acids long are poor immunogens. In general, to increase its immunogenicity, the presentation of the peptide should be improved, and molecular weight needs to be increased. Many attempts have been made to couple peptide immunogens to different carrier proteins [e.g. keyhole limpet haemocyanin (KLH) or ovalbumin]. This leads to very complex structures, however. We used a controlled conjugation of a peptide to a single long-chain fatty acid like palmitic acid by a thioester or an amide bond. It was found that these S-palmitoylated peptides were much more immunogenic than N-palmitoylated peptides and at least similar to KLH-conjugated peptides with respect to appearance and magnitude of induced antibodies (canine parvovirus) or immunocastration effect (gonadotropin-releasing hormone). For chemical synthesis of thioesters, we established conditions for solution and solid-phase synthesis. In both phases, Cys(SBut) could only be deprotected efficiently using phosphines, and S-acylation was accomplished using standard coupling at pH 5. We speculate that, in vivo, the presence of an appropriate fatty acid chain, chemically linked through a labile thioester bond, greatly enhances immunogenicity, because it represents a favourable substrate for cleavage by cellular thioesterases in cells of the immune system.

In vitro inhibition of the biological activity of follicle-stimulating hormone by anti-peptide antisera representing the human follicle-stimulating hormone beta subunit sequence 33-53.

There are few male contraceptive methods, and research is required to broaden the scope of available male antifertility methods. Two approaches toward hormonal contraception are currently being investigated. The first relies on elimination of testosterone while the second is based upon immunizations against FSH. However, most anti-whole FSH antisera cross-react with LH, thereby possibly inhibiting testosterone and leading to potential loss of libido. Therefore, a more effective alternative would be to define an FSH peptide that differs significantly from LH in order to prevent cross-reactivity between anti-FSH antisera and LH. Two peptides were selected from the beta subunit of FSH that were considered to be inducers of anti-FSH activity but not anti-LH activity. The first peptide (sequence beta33-53) is a linear antigenic site of human FSH found only in anti-FSH antisera that do not cross-react with LH. The second peptide (sequence beta81-95) is a part of FSH that confers receptor specificity. These peptides, in monomer and tandem form, were used to immunize rabbits. The antisera were tested for inhibition of FSH activity in a bioassay; they were also tested in a Leydig cell assay to detect anti-LH activity. It was found that antisera raised against the beta33-53 tandem could inhibit the FSH bioactivity but not that of LH. Antisera against the beta33-53 monomer or the beta81-95 monomer or tandem did not inhibit FSH. Thus, the tandem peptide beta33-53 is an attractive candidate for use as antigen in a male contraceptive vaccine. The better results obtained with tandem vaccinations might be related to the ability of the tandem peptide to direct the antibody response toward the N-terminal end of the peptide and to raise antisera with the ability to react with shorter chains of amino acids.

Screening of a small set of random peptides: a new strategy to identify synthetic peptides that mimic epitopes.

Small diversity libraries, composed of 4550 synthetic dodecapeptides and 8000 synthetic tripeptides, have been used to identify sequences homologous to small linear and non-linear parts of epitopes. Here we report that synthetic peptides identified through alignment of dodecapeptides and tripeptides derived from these small libraries have, in direct ELISA and/or competitive ELISA, activities similar to that of peptides covering the native epitope and similar to that of peptides derived from large expression libraries composed of 10(6)-10(7) random peptides. This result was obtained with the monoclonal antibodies 6A.A6 and M2. Mab 6A.A6 binds the transmissible gastroenteritis virus (TGEV) and mAb M2 binds the FLAG-peptide, an affinity tag. It was also found that the antibody binding activity of peptides, derived from small or large libraries, can strongly depend on the way in which the peptide is presented to the antibody, i.e. high antibody titers were obtained when these peptides were synthesized on pins or coated onto microtiter plates, whereas low IC50s were obtained with these peptides in solution. We postulate that small peptide libraries may be a powerful tool to quickly identify new peptides that can be used as sensitive markers for mAbs of interest.

Solution structure of the immunodominant region of protein G of bovine respiratory syncytial virus.

The three-dimensional solution structure of the immunodominant central conserved region of the attachment protein G (BRSV-G) of bovine respiratory syncytial virus has been determined by nuclear magnetic resonance (NMR) spectroscopy. In the 32-residue peptide studied, 19 residues form a small rigid core composed of two short helices, connected by a type I' turn, and linked by two disulfide bridges. This unique fold is among the smallest stable tertiary structures known and could therefore serve as an ideal building block for the design of de novo proteins and as a test case for modeling studies. A characteristic hydrophobic pocket, lined by conserved residues, lies at the surface of the peptide and may play a role in receptor binding. This work provides a structural basis for further peptide vaccine development against the severe diseases associated with the respiratory syncytial viruses in both cattle and man.