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This paper reviews how immunomodulation through active vaccination has evolved in the past 25 years. Although initially it progressed isolated from the main stream of immunological research and vaccine development, lately it merged with this main stream and is taking full advantage of the newest developments in vaccinology. The first immunomodulation vaccine is already on the market, while various others are close to it. Not in the least because one of the major stumbling blocks of immunomodulation through active vaccination, the inherent low immunogenicity of ‘self’ antigens, has in a number of other cases been solved. Most progress has been made in veterinary applications and has helped to formulate practical rules, necessary to break immunotolerance. It is not unlikely that these rules will be used to design better immunomodulation vaccines to be used in humans; notably to control fertility or combat tumours.

Immunization against gonadotrophin releasing hormone (GnRH) was studied as an alternative for the commonly used surgical castration in stallions. Two GnRH vaccines comprising non-mineral oil adjuvants were evaluated for their potential to induce high antibody titers directed against GnRH and subsequent effects on reproductive characteristics. Twelve sexually mature male hemicastrated Shetland ponies were assigned to three groups. Group 1 and 2 were injected with 1mg peptide equivalent of G6k-GnRH-tandem-dimer conjugated to ovalbumin (OVA) in CoVaccine HT adjuvant (GnRH/CoVaccine) and in Carbopol (GnRH/Carbopol), respectively, and group 3 was injected with CoVaccine HT adjuvant without antigen (controls). After immunization no adverse effects were observed with respect to the injections sites or general health. Two weeks after the second vaccination antibody titers against GnRH increased rapidly in all animals of the GnRH/CoVaccine group, at the same time reducing serum testosterone levels maximally for the further duration of the experiment. In the GnRH/Carbopol group antibody responses and effects on testosterone levels were intermediate in two stallions and not apparent in the remaining stallions of this group. Semen evaluation showed that from 2 weeks after the second immunization onwards, sperm motility was affected in all stallions treated with GnRH/CoVaccine and one stallion treated with GnRH/Carbopol. Seven weeks after the second immunization, no semen could be collected from two stallions, one of each group, due to suppressed libido. Histological examination of the testes, 15 weeks after the initial immunization, demonstrated reduction in seminiferous tubuli diameters in all stallions of the GnRH/CoVaccine group and one stallion of the GnRH/Carbopol group. Furthermore, spermatogenesis was extremely disorganized in these stallions, as indicated by absence of the lumen in the seminiferous tubules, the absence of spermatozoa and spermatids in the tubular cross-sections and the impossibility to determine the stage of the tubular cross-sections. Testis size was also substantially reduced in three out of four stallions treated with GnRH/CoVaccine. The results demonstrate that two immunizations with G6k-GnRH-tandem-dimer-OVA conjugate in a suitable adjuvant such as CoVaccine HT caused a rapid and complete reduction of serum testosterone levels in sexually mature stallions, subsequently leading to reduced sperm motility and affected testis function, while no adverse reactions were observed after immunizations.

Protein transduction domains (PTDs) have proven to be an invaluable tool to transduce a wide variety of cargoes across the plasma membrane and into intact tissue including brain. The PTDs are able to deliver biologically active molecules both in vitro and in vivo. To exploit the full potency of transport peptides it is important to know the efficiency, what kind of cargoes can be transported and for what application it can be used. Because fixation artifacts and irreversible membrane binding may cause misinterpretation of the amount of internalization of polybasic peptides, we have developed an enzyme transduction assay based on the intracellular loading of a cell permeable substrate. In this assay, a fluorescent signal is generated by internalized enzyme in intact cells and not by membrane-bound or extracellular enzyme. This assay demonstrates the true efficiency of transport peptides. To check whether the efficiency is sufficient for intracellular applications we demonstrated the internalization of an enzyme substrate in the cell and the replacement of an enzyme in a deficient cell. Comparison of many different polybasic PTDs showed that the efficiency of transport was dependent on the type of cargo. Therefore, the unique characteristic of a PTD may only become apparent when it is selected for a particular application. Rules for optimisation of PTDs for particular applications are now emerging and opening the way for a new generation of drug delivery agents.

Protein transduction domains (PTDs) have proven to be an invaluable tool to transduce a wide variety of cargo's including peptides across the plasma membrane and into intact tissue. The PTDs are able to deliver biologically active molecules both in vitro and in vivo. This study describes many new polybasic PTDs of which some are just as potent as the PTDs derived from extracellular RNAses or other published PTDs. Large differences in potency became apparent when the PTDs are coupled to particular cargoes. Therefore, the unique characteristic of a PTD may only become apparent when it is selected for a particular application. Rules for optimization of PTDs for particular applications are now emerging and open the way for a new generation of drug delivery agents. Because fixation artifacts and irreversible membrane binding may cause misinterpretation of the amount of internalization of polybasic peptides, we have developed an enzyme transduction assay based on the intracellular loading of a cell permeable substrate. In this assay, a fluorescent signal is generated by internalized enzyme in intact cells and not by membrane-bound or extracellular enzyme.

In this study, the performance of male pigs immunized against GnRH was determined in relation to the onset of their biological response to the immunization. Pigs were immunized at 9 and 17 wk of age and were housed in a pen together with both a surgically castrated and an intact boar littermate. Feed intake was restricted to 2.8 to 3.2 times maintenance requirement for energy. Animals were weighed weekly and slaughtered at 108 kg BW. Depending on the time of onset of the response after immunization in terms of biological effects, immunized pigs were retrospectively grouped into two categories. One category consisted of the immunized pigs, which had undetectable or low levels of LH and testosterone at the time of booster immunization-known as "early" responding immunocastrates (E-IM, n = 8), whereas the "late" responding immunocastrates (L-IM, n = 7) had substantial LH and testosterone levels at that time. This dichotomy of the response to immunization also was reflected in testis weight, with 17 g and 40 g for E-IM and L-IM pigs, respectively. At slaughter, testis size and weight were reduced (P < 0.001) in the immunocastrated pigs as compared to the intact boars. Androstenone concentrations in backfat of all immunocastrated pigs were undetectable. Growth performance (i.e., ADG and feed efficiency [FE, g gain/kg feed]), was better in boars and L-IM pigs than in surgical castrates and E-IM pigs (P < 0.05). Average daily gain and FE did not differ between E-IM pigs and the surgical castrates, but intact boars performed better than L-IM (P < 0.02). There were no significant differences in carcass quality (backfat thickness and meat percentage) between boars and surgical castrates at slaughter. However, for both characteristics L-IM pigs and intact boars performed better (P < 0.03) than E-IM pigs. Thus, growth performance in L-IM is better than in either E-IM or surgical castrates.

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 C16-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.

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.

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.

Superovulation treatment of cows can benefit from the application of very pure recombinant bovine FSH (rbFSH), which is produced in nonmammalian cells. rbFSH is completely free of LH, and therefore can possibly reduce the variability in the results of superovulation. Furthermore, it does not contain brain-tissue-derived proteins and, when produced under serum-free conditions, it is free of other mammalian substances or potentially infectious material. We have produced rbFSH in insect cells, with the ultimate aim of inducing superovulation in cattle. Sf21 insect cells were coinfected with two recombinant baculoviruses, containing the cDNAs of bovine FSH alpha- and beta-subunits respectively. High levels of production of bioactive rbFSH were obtained after cloning cDNA that contained a major part of the 3' untranslated region of the bFSH beta gene. Maximum production of rbFSH 1-5 micrograms/ml (as measured by immunoassay) was obtained 70-90 h after infection. The recombinant material was highly potent in two in vitro bioassays, giving biological activities of 13 IU/ml (Y1 cell rounding assay), 22 IU/ml (Y1 cell cAMP assay), and 23 IU/ml (bovine oocyte maturation inhibition assay), and had a lower but significant activity of 6 IU/ml in the rat Sertoli cell assay. rbFSH was purified by immunoaffinity chromatography, using a monoclonal antibody directed against the human FSH beta-subunit. The purified heterodimer appeared to be homogeneous by SDS-PAGE, whereas the free beta-subunit appeared as a doublet, possibly indicating differently glycosylated forms. Intact heterodimer and both subunits were further identified by western blot analysis, and showed apparent molecular masses of 20 kDa (alpha-subunit), 23 kDa (beta-subunit) and 32.5 kDa (heterodimer). This insect-cell-produced rbFSH did not bind to wheat germ agglutinin, thus indicating that glycosidic side-chains may not contain terminal sialic acid. The relevance of a large 3' untranslated region in bFSH beta cDNA to the level of production of rbFSH, and the possible implications of the pattern of glycosylation for the biological activity of the recombinant hormone are discussed.

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 β subunit of FSH that were considered to be inducers of anti-FSH activity but not anti-LH activity. The first peptide (sequence β33-53) 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 β81-95) 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 β33-53 tandem could inhibit the FSH bioactivity but not that of LH. Antisera against the β33-53 monomer or the β81-95 monomer or tandem did not inhibit FSH. Thus, the tandem peptide β33-53 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.

There are currently two major approaches to hormonal male contraception. One relies on testosterone (analogs) either alone or in combination with gonadotropin releasing hormone (GnRH) (analogs or immunizations), the other on immunizations against follicle-stimulating hormone (FSH). Theoretically, the latter method will suppress spermatogenesis whilst not interfering with libido. An absolute requirement is, however, that an anti-FSH vaccine does not induce anti-luteinizing hormone (LH) antibodies (LH being responsible for the induction of testosterone which is necessary-to maintain libido). In this report we show that when whole FSH is used for vaccination, in most cases in addition to biological activity against FSH, anti-LH activity is also induced. By systematic analysis of the antisera raised with FSH using systematic epitope scanning (PEPSCAN) we found differences between the FSH-specific and FSH-nonspecific sera. Only the FSH-specific antiserum contained antibodies that recognized amino acid sequence 37–55 on the β-subunit in a linear manner. Because antibodies against this epitope have not been found in the cross-reactive sera this epitope forms a prime candidate for an anti-FSH contraceptive vaccine

The rational design of effective oral vaccines based on synthetic peptides is a very ambitious undertaking, and involves the solution of huge problems related to protection of the antigens against degradation in the alimentary tract, efficient uptake of the antigens by the relevant cells, and efficient induction of long lasting systemic immunity, local immunity, or both. This paper summarises the steps, necessary to develop such synthetic oral vaccines. These steps involve: (1) the definition of B-cell epitopes; (2) the definition of T-cell epitopes; (3) definition of the carrier or backbone molecule; (4) definition of an immunomodulating element; (5) definition of an adjuvant element; and (6) definition of a targeting element. Good progress is being made with respect to the first three steps, the other steps still provide major challenges, notably the definition of targeting elements. Nevertheless, the first synthetic oral vaccines may become reality in the near future, depending on the speed by which new technology in the area of molecular recognition will develop, i.e. the appropriate chemistry, organic chemistry, molecular modelling, resolution of the molecular interaction of key molecules in microbiology and immunology.

Epitopes on thaumatin and monellin were studied using the PEPSCAN-technology. The antibodies used were raised against thaumatin. Only antibodies that, in an ELISA, both recognized thaumatin and monellin were used in the PEPSCAN-analyses. On thaumatin two major overlapping epitopes were identified. On monellin no epitopes could be identified. The identified epitope region on thaumatin shares structural features with various peptide and protein sweeteners. It contains an aspartame-like site which is formed by Asp21 and Phe80, tips of the two extruding loops KGDAALDAGGR19-29 and CKRFGRPP77-84, which are spatially positioned next to each other. Furthermore, sub-sequences of the KGDAALDAGGR19-29 loop are similar to peptide-sweeteners such as L-Asp-D-Ala-L-Ala-methyl ester and L-Asp-D-Ala-Gly-methyl ester. Since the aspartame-like Asp21-Phe80 site and the peptide-sweetener-like sequences are also not present in non-sweet thaumatin-like proteins it is postulated that the KGDAALDAGGR19-29- and CKRFGRPP77-84 loop contain important sweet-taste determinants. This region has previously not been implicated as a sweet-taste determinant of thaumatin.

Immunization against GnRH-like peptides to immunocastrate boars can be very effective in preventing boar taint and in reducing testis weight (TW) and size (TS). Experiments using vaccines with different efficacies resulted in a total of 203 male pigs with widely diverging TW and TS. The relations of the latter parameters with androstenone (AN) showed that AN levels were always lower than 0.5 μg/g (the limit for the sensory detectability of boar taint) when TW was

In the present study the inhibitory effects of a panel of 21 monoclonal antibodies (moabs) to rat interleukin 1 beta (rIL-1 beta) on the binding of 125I-labeled rIL-1 beta to murine type I IL-1 receptors on EL4 cells were investigated. Furthermore, the epitopes of these moabs were determined by the use of the pepscan technique, and these epitopes were visualized on a three-dimensional model of rIL-1 beta. Some moabs (SILK 3, 4, 5, 6, and 22) inhibited receptor binding of radioiodinated rIL-1 beta at concentrations that are similar to the dissociation constant values of antibody-rIL-1 beta binding. Another group of moabs (SILK 7, 11, 20, 21, and 23) also inhibited receptor binding but only at concentrations that are 10-150 times higher than their dissociation constants. A large group of moabs did not affect receptor binding in the concentration range tested, and two moabs enhanced the binding of rIL-1 beta to type I receptors. The result of pepscan analysis shows that the moabs bound to one or more of the amino acid sequences 35-49, 66-85, 78-97, 106-124, and 123-143 of mature rIL-1 beta. Modeling of rIL-1 beta shows that the binding domains of SILK 4, 5, 6, and 22 (sequence 123-143) is located at the closed end of the molecule, indicating that this part of rIL-1 beta harbors domains that are crucial for type I receptor binding. The binding domain of SILK 3 (sequence 66-85) is also located at this end of the molecule. In contrast, the binding domains of SILK 7, 11, 20, 21, and 23 (sequence 78-97) are located at the open end of the molecule, which is at the same face as the amino- and carboxy-terminals. The binding domain of SILK 16 (sequence 106-124) is positioned at the center of the molecule. It is concluded that the closed end of rIL-1 beta contains sequences that are crucial for its binding to type I receptors on murine EL4 cells. Because of the high concentrations of antibodies to residues 78-97 of rIL-1 beta that are needed to interfere with receptor binding, the importance of these domains in binding to type I receptors remains uncertain.

Immunomodulation of reproductive systems is a means of increasing or decreasing reproductive efficiency. In veterinary medicine, it may be applied to ‘castrate’ animals without removing the gonads and to treat diseases caused by hormonal imbalance. Endogenous hormones are recognized as ‘self’ by an animal. To acquire a foreign antigen for immunization purposes, endogenous hormones are usually coupled to a molecule that is foreign to the animal. For immunoneutralization of endogenous hormones, a high titer of specific antibodies is required, a requirement that is often difficult to meet. Nevertheless, in the last decade it has been demonstrated that endocrine systems can be influenced using active and passive immunization against steroid hormones, such as androstenedione, protein hormones, such as inhibin, and peptide hormones, such as luteinizing hormone releasing hormone (LHRH). A new and promising development is the use of peptides as antigens in active immunization against hormones such as inhibin and human choriogonadotrophin.

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 (P0.05) 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 (P0.05) in immunocastrates than in intact controls (9.4 +/- 1.1 and 390.9 +/- 67.2 g, respectively). Antibody titers were significantly lower (P0.05) 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.

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

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.

Synthetic peptides representing relevant B-cell epitopes are, potentially, ideal antigens to be used in diagnostic assays because of their superior properties with respect to quality control as compared to those of biologically derived molecules and the much higher specificity that sometimes can be obtained. It is thus not a surprise than an increasing number of synthetic peptide based diagnostic assays are being developed or already are commercially available. In this report methods to define useful synthetic peptides, and diagnostic systems that are under development or on the market are discussed.

The FLAG peptides DYKDDDDK and MDYKDDDDK are widely used affinity tags. Here we describe new variants of the FLAG peptides which, in direct ELISA, showed selective and differential binding to the commercially available anti-FLAG monoclonal antibodies M1, M2 and M5. Variants of the FLAG peptides were synthesized on polymer-grafted plastic pins, and in an ELISA incubated with mAbs M1, M2 and M5. Among the newly identified tag sequences are those that bind only one of the anti-FLAG mAbs and those that bind only two or all three of the anti-FLAG mAbs. Examples of new tag sequences are MDFKDDDDK (which binds mAb M5 and does not bind mAbs M1 and M2) and MDYKAFDNL (which binds mAb M2 and does not bind mAbs M1 and M5). The sensitivity in direct ELISA of some variants was increased, e.g. using mAb M2 it was found that replacing DDDDK in MDYKDDDDK by AFDNL increased the sensitivity in ELISA at least 10-fold. The activity of this peptide was studied in more detail. In different direct ELISAs, in which MDYKAFDNL was synthesized on polyethylene pins, coated onto polystyrene microtiter plates or onto nitrocellulose paper, the activity of this peptide was similar, i.e. increased at least 10-fold over that of MDYKDDDDK. Remarkably, in competitive ELISA the binding activity of soluble MDYKAFDNL was decreased 10-fold over those of soluble MDYKDDDDK or DYKDDDDK. The results seem to suggest that, in solution, the conformation of MDYKAFDNL is more 'unstructured' compared to its conformation when coated or linked to a carrier. We postulate that the newly described tag sequences may be used as affinity tags to separately detect, quantify and purify multiple co-expressed proteins and/or subunits.

The terminals of the hypothalamic gonadotrophin hormone-releasing hormone (GnRH) neurons are located within the median eminance and thereby extend beyonf the protection of the blood-brain barrier. Thus, these terminals may be subjected to direct autoimmune action in animals that are actively immunised against GnRH. Boars (male pigs) (n = 108) were actively immunised against GnRH by two successive injections with synthetic GnRH, covalently coupled to KLH and dissolved in CFA or IFA. They were killed at 26 weeks of age. Immunised boars were selected on the basis of the resultant testes size, which indicates the effectiveness of the immunisation. The hypothalami of 25 selected animals were studied by histological and immunocytochemical techniques and compared with the hypothalami of three sham- and nine control animals. In the immunised animals, changes in the GnRH system had taken place. These comprised dystrophy of the perikarya and a sharp decrease of the GnRH immunocytochemical reactivity in the terminals within the median eminence. In addition, various degrees of inflammatory reactions were present, particularly within the median eminence. These consisted of tissue disruption by edema, collapse of the capillaries, fibrosis and infiltration with fibroblasts. In addition, accumulations of neurosecretum within the median eminence in combination with hypertrophy of magnocellular neurons within the hypothalamus were present. The reactions were restricted to the median eminence and did not involve other neurohemal organs or other parts of the hypothalamus. A correlation could be established between the incidence of the lesions and the effectiveness of the GnRH autoimmunity (as indicated by the size and endocrine funtion of the gonads and the anti-GnRH titres). Changes in extra- and intracellular IgG immunocytochemical reactivity within the median eminence indicated the involvement of IgG. The effects were absent from control and sham vaccinated animals and after vaccinations with other compositions of the vaccine. Thus, hypothalamic lesions have been observed in this selected group of animals, vaccinated against GnRH with this particular vaccine.

Follicle-stimulating hormone (follitropin, FSH) belongs to a group of closely related glycoprotein hormones that contain two noncovalently linked dissimilar subunits designated alpha and beta. By using synthetic peptides, several receptor interaction sites in these hormones have been identified; however, the peptides have a reduced potency (lowest effective concentration of 10(-4) to 10(-5) M) relative to the hormone itself (10(-8) to 10(-11) M). This suggests that the peptides represent only a portion of a larger recognition site in the intact hormone that comprises parts of both the beta and the alpha chains. To develop peptides that exhibit FSH-antagonistic activity at low concentrations, we have constructed a three-dimensional model for FSH, which is based on an alignment of both the beta and the alpha chains of glycoprotein hormones with thioredoxin, for which x-ray diffraction data are available. This model resulted in the prediction of a conformational receptor-binding site in FSH, in which (parts of) three earlier proposed binding regions on the FSH molecule [namely, the regions FSH alpha-(34-37), with the amino acid sequence SRAY; FSH beta-(40-43), with the amino acid sequence TRDL; and FSH beta-(87-94), the "determinant loop" with the amino acid sequence CDSDSTDC] are located within 10 A of one another. On the basis of this model, peptides have been synthesized in which two of these binding regions are linked by a synthetic amino acid whose length was derived from the model, Ac-TDSDS-NH-(CH2)5-CO-SRAY-NH2 and Ac-SRAY-NH-(CH2)4-CO-TRDL-NH2. Both peptides inhibited FSH-induced cAMP production in Sertoli cells at 1000-fold lower concentrations (10(-7) M) than the peptides Ac-TRDL-NH2, Ac-SRAY-NH2, or Ac-TDSDS-NH2. In another peptide, Ac-TDSDS-NH-(CH2)5-CO-SRAY-NH-(CH2)4-CO-TRDL-NH2, all three binding regions have been linked. This peptide appeared to be a strong agonist of FSH action, as measured by the ability to stimulate cAMP production, at concentrations as low as 10(-7) M. The observation that a synthetic peptide, in which (parts of) three earlier described receptor interaction sites are combined according to the three-dimensional model, can mimic the action of FSH, at 10(-7) M, shows that this model is useful to predict a conformational receptor-binding site in FSH and that combination of only a few amino acid residues from the alpha and beta chains of FSH in a small synthetic peptide is sufficient to transduce a signal upon binding to the receptor.

The initiation and maintenance of spermatogenesis are under the control of follicle stimulating hormone (FSH) and testosterone. For different mammalian species, it is clear that FSH is an important regulator, in particular during the first wave of spermatogenesis, and that testosterone is the major stimulator of spermatogenesis in adult males. FSH exerts multiple effects on its target cell, the Sertoli cell, and its action is most evident in testis from immature or hypophysectomized animals (Fritz 1978). However, at all stages of development, the actions of FSH and testosterone seem to be highly integrated. Although the importance of FSH in adult males is not clear, in either the human or in animal species, it appears that FSH is necessary for quantitative maintenance and repair of spermatogenesis. It has been shown that the FSH receptor is carefully regulated in adult rats and, presumably, has an important function in spermatogenesis (Heckert and Griswold 1992). A marked quantitative stimulation of spermatogenesis occurs when FSH is administered to intact adult cynomolgous monkeys (Van Alphen et al. 1988). Administration of anti-FSH antibodies to rats leads to a decrease in spermatogonia (Vaishnav and Moudgal 1992). It cannot be excluded that male fertility will be impaired upon inhibition of FSH action. The possibility of inducing male infertility by blocking FSH action is very attractive for two reasons. First, according to current knowledge, testicular Sertoli cells are the exclusive target cells for FSH in the male, which guarantees specificity. Second, lutropin (LH) action and testicular androgen production would not be affected. Different approaches have been considered to interfere with FSH stimulation of the testis, without acting upon LH stimulation of testicular androgen production. In our laboratory, we are trying to develop FSH antagonistic peptides and peptide vaccines directed against FSH. Synthetic peptides and especially synthetic peptide vaccines instead of “native” protein hormone (vaccines) have been predicted to be especially well suited for mass application in developing countries because peptides are relatively cheap, easy to produce, to store, to transport and to modify. However, a major obstacle to the development of anti-FSH vaccines or FSH antagonistic peptides has been the absence of detailed knowledge about the antigenic determinants and receptor sites on the hormone.

Antibodies were raised against synthetic peptides of two regions of the surface protein VP1 of foot-and-mouth disease virus. The peptides were conjugated with keyhole limpet hemocyanin via C- or N-terminal amino acid residues by use of different coupling agents. The fine specificity of the resulting antibodies was determined by PEPSCAN methods. In general, amino acid residues specific for antibody recognition tended to be located opposite to those used for coupling with the carrier protein. Depending on the method of conjugation, the orientation of the peptide at the carrier protein changes and directs the immune response. Thus, the method of conjugation can be used to manipulate the immune response and to improve the antiviral activity of antipeptide antibodies. The PEPSCAN method is an effective monitor in this process.

Analyse de l'epitope de la proteine de surface de 25 kD de P. falciparum (pfs 25) sur lequel se fixe l'anticorps monoclonal 32F81 (AM32F81) qui bloque la transmission du parasite. Synthese de nonapeptides a sequences chevauchantes determinees a partir de la sequence de pFs25 et etude de leur reactivite avec AM32F81. Sur le nonapeptide qui donne la plus forte reactivite, recherche par substitution d'acide amine des acides amines impliques dans l'interaction specifique avec AM32F81