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Summary The immune response upon vaccination and revaccination of cattle with a vaccine against FMD type A was analysed. Neutralizing antibodies were measured and antibodies precipitating with homologous complete virus, trypsin treated virus, 12 S viral sub-units and with heterologous virus type C were assayed by radial immuno diffusion, both in whole sera and in sera fractionated over Sephadex G 200. The results show the development of neutralizing activity and precipitating activity against the complete and the trypsin treated virus in both the 19 S and 7 S serum fractions. Precipitating activity against 12 S viral subunits was found in the 7 S serum fractions, whereas the precipitating activity against the heterologous (type C) virus was only observed in the 19 S serum fractions. After revaccination the neutralizing ability of a given quantitiy of precipitating antibodies increased. In the course of the primary response the proportion of antibodies directed to the 12 S virus sub-units as compared to that directed to the complete virus increased slightly. In contrast the ratio of antibodies directed to trypsin treated virus to antibodies directed to complete virus hardly changed. In the 19 S serum fraction only part of the precipitating activity seems to be due to the vaccination. This also applied to the activity against heterologous type C virus. Zusammenfassung Antikorperreaktion gegen Maul- und Klauenseuche-Virus (FMDV) Teil I: Die Reaktionen in Seren von vakzinierten Tieren mit komplettem Virus, Trypsin-behandeltem Virus 12S-Virusuntereinheiten und heterologem Virus Es wurde die Immunreaktion auf die Vakzination und Revakzination von Rindern mit einer Vakzine gegen MKS-Typ A analysiert. Die neutralisierenden Antikorper wurden gemessen, sowie die mit homologem komplettem Virus, Trypsin-behandeltem Virus, 12 S-Virusuntereinheiten und mit heterologem Typ C-Virus prazipitierendem Antikorper mit Hilfe der Radial-Immunodiffusion, einmal an den Gesamtseren und andererseits an uber Sephadex G 200 fraktionierten Seren bestimmt. Die Ergebnisse zeigen die Entwicklung neutralisierender und prazipitierender Aktivitat gegen komplettes und Trypsin-behandeltes Virus sowohl in der 19 S- als auch in der 7 S-Serumfraktion. Prazipitierende Aktivitat wurde auch in der 7 S-Fraktion gegen die 12 S-Virusuntereinheiten nachgewiesen, wogegen sie gegen heterologes Typ C-Virus nur in der 19 S-Serumfraktion beobachtet werden konnte. Nach der Revakzination stieg die Neutralisationskraft einer vorhandenen Menge prazipitierender Antikorper an. Im Verlauf der primaren Antikorperreaktion stiegen die Antikorper gegen die 12 S-Virusuntereinheiten im Verhaltnis zu denen gegen das komplette Virus etwas starker an. Dagegen anderte sich das Verhaltnis zwischen den Antikorper gegen Trypsin-behandeltes Virus und denen gegen komplettes Virus kaum. In der 19 S-Serumfraktion scheint nur ein Teil der prazipitierenden Aktivitat auf die Vakzination zuruckfuhrbar zu sein. Dies trifft auch fur die Aktivitat gegen heterologes Typ C-Virus zu. Resume Reaction des anticorps contre virus de la fievre aphteuse (FMDV) Premiere partie: Les reactions dans des serums d'animaux vaccines avec un virus complet, un virus trypsinise, de sous-unites virales 12 S et un virus heterologue La reaction immunitaire a une vaccination et a une revaccination de bovins avec un vaccin anti-aphteux du type A a ete analysee. On a mesure les anticorps neutralisants ainsi que les anticorps precipitants a l'aide de l'immunodiffusion radiale avec des serums complets et des serums fractionnes sur Sephadex G 200 pour un virus homologue complet, un virus trypsinise, des sous-unites virales 12 S et un virus heterologue du type C. Les resultats montrent le developpement d'une activite neutralisante et precipitante contre un virus complet et trypsinise dans la fraction serique 19 S et 7 S. Une activite precipitante a egalement ete demontree dans la fraction 7 S vis-a-vis de sous-unites virales 12 S, mais n'a pu etre observee que dans la fraction 19 S pour le virus heterologue du type C. La force de neutralisation s'est augmentee d'une quantite presente d'anticorps precipitants apres le revaccination. Au cours de la reaction primaire des anticorps, les anticorps contre les sous-unites virales 12 S sont montes un peu plus fort que ceux contre l'anticorps complet. Le rapport entre les anticorps anti-virus trypsinise et antivirus complet fut a peine different. Une partie seulement de l'activite precipitante dans la fraction serique 19 S semble etre due a la vaccination. Ceci est egalement valable pour l'activite vis-a-vis du virus heterologue du type C. Resumen Reaccion de los anticuerpos frente al virus aftoso (FMDV) Parte Io: Las reacciones en sueros sanguineos de animales vacunados con virus completo, virus tratado con tripsina, subunidades virosicas 12 S y virus heterologo Se analizo la reaccion inmunologica a la vacunacion y revacunacion de bovinos con una vacuna antiaftosa del tipo A. Se midieron los anticuerpos neutralizantes, asi como los anticuerpos precipitantes con virus homologo completo, virus tratado con tripsina, subunidades virosicas 12 S y el virus heterologo tipo C, con ayuda de la inmunodifusion radial, valorandose una vez en los sueros totales y otra en los sueros fraccionados sobre Sephadex G 200. Los resultados evidencian el desarrollo de la actividad neutralizante y precipitante frente al virus completo y tratado con tripsina tanto en la fraccion serica 19 S como en la 7 S. Actividad precipitante se identifico tambien en la fraccion 7 S frente a las subunidades virosicas 12 S, mientras que frente al virus heterologo tipo C se pudo apreciar solo en la fraccion serica 19 S. Tras la revacunacion ascendio la potencia de neutralizacion de una cantidad presente de anticuerpos precipitantes. En el curso de la reaccion primaria de anticuerpos aumentaron un poco mas los anticuerpos frente a las subunidades virosicas 12 S con relacion a aquellas frente al virus completo. Por el contrario, apenas se modifico la relacion entre los anticuerpos frente al virus tratado con tripsina y los mismos frente al virus completo. Parece ser que en la fraccion serica 19 S solo se puede atribuir una parte de la actividad precipitante a la vacunacion. Esto tambien reza con la actividad frente al virus heterologo tipo C.

Due to the advantageous properties of synthetic molecules compared to biological ones biological molecules in diagnostic tests are replaced increasingly by synthetic ones, usually synthetic peptides or related molecules. The replacement of biological antigens by synthetic peptides is most advanced at present, as well as the use of site-specific antibodies induced with synthetic peptides. Moreover recent results indicate that synthetic molecules may also replace antibodies. Ultimately this will lead to diagnostic assays built of synthetic molecules only.

Three rabbit antibodies (R521, R505, R524) were produced, and raised to synthetic peptides corresponding to residues 94-105, 100-111, and 223-234, respectively, of the sheep prion protein (PrP). Epitope mapping analysis revealed the monospecific character of antisera R505 and R524. In addition to the amino acid sequence against which it was raised, R521 also recognized Other small epitopes. ELISA and radio-immunoprecipitation were used to assess the relative immunoreactivities of the antisera to the normal sheep prion protein (PrP(c)). Highest reactivity was found for R521, followed by R505 and R524. According to Western blot analysis, all three sera specifically reacted with the prion proteins PrP(sc) and PrP27-30, extracted from the brain stem of a scrapie-affected sheep. Yet, with R505 not all of the lower molecular weight deglycosylated forms could be detected. Contrary to the immunoreactivities found with the PrP(sc) and PrP27-30 isoforms, only R521 recognised PrP(c) from a healthy sheep. The usefulness of all three anti-peptide sera in the immunohistochemical detection of PrP(sc) in brain stem and tonsils of scrapie-affected sheep was demonstrated and compared with an established rabbit anti-PrP serum. (C) 2000 Wiley-Liss, Inc.

Two different epitope mapping techniques were used to identify linear epitopes recognised by polyclonal IgG antibodies from rabbits immunised with bovine beta lactoglobulin (BLG), which is generally regarded as a major allergen in milk. The first, PEPSCAN, was used to investigate the binding of several rabbit polyclonal antisera to sequential overlapping peptides (12-mers) across the sequence of BLG. Each peptide was synthesized on a different polypropylene PIN, and a standard ELISA procedure was used to locate which of these peptides bound the antibodies under investigation. Comparisons of PEPSCANs for antisera from six different rabbits showed that each rabbit recognized a similar set of epitopes within BLG. PEPSCAN analysis also showed that polyclonal antibodies from the mouse recognize a set of epitopes similar to those recognized by the rabbit. The second epitope mapping technique is known as phage display and utilizes libraries of randomized short peptides fused to the coat proteins of filamentous phage as a source of epitopes for analysis. A gene VIII phage display library was used in this study with constrained nonapeptides, which were screened for epitopes recognized by affinity purified rabbit anti-BLG IgG. Immobilised rabbit anti-BLG IgG was screened in two separate experiments, each consisting of three rounds of panning. For each separate experiment, a sensitive phage ELISA was used to screen several hundred single phage clones for binding to anti-BLG IgG immobilised on microtiter plates. As a result, a number of positive phage were identified from the two separate screens of the library (19 different peptides were isolated, which resembled four different regions of BLG). The identified sequences were found to constitute a subset of the linear epitopes recognized by the PEPSCAN technique. The coordinates of the crystal structure of BLG were used to display mapped epitopes on its structure. This study has permitted detailed mapping of the major linear antigenic regions within BLG recognised by IgG antibodies from immunised rabbits and mice.

By combining DNA subclone and synthetic peptide approaches, immunogenic epitopes have been mapped from the mycobacterial heat shock proteins (HSP) of 18, 65 and 70 kD, as well as from a novel non-HSP antigen of 24 kD recognized by antigen-specific human T cells. In addition the HLA-DR molecules used in antigen presentation of the individual peptide-defined epitopes have been identified. The donor groups used for establishing antigen-specific CD4+ T cell clones and lines were primarily healthy subjects immunized with Mycobacterium bovis BCG and killed Mycobacterium leprae. The results showed that HSP18 contains one epitope (aa 38-50) presented by HLA-DR4; HSP65 contains 4 epitopes presented by DR1 and DR2 and 5 epitopes presented by DR4; whereas HSP70 contains multiple epitopes presented by DR2, DR3, DR5, DR7, and DRw53. The 24-kD non-HSP antigen also displayed one HLA-DRw53-restricted T cell epitope. In conclusion, the individual HSP T cell epitopes defined here are exclusively presented by only one HLA-DR molecule. However, at the protein level the results suggest that HSP65 and HSP70 are relevant to the subunit vaccine design, since they contain multiple T cell epitopes which can be presented by a spectrum of different HLA-DR molecules.

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.

The p190 protein (also called MSA1 or MSP1) of the asexual blood stage forms of Plasmodium falciparum, a human malaria vaccine candidate, shows polymorphism between different isolates. Mice were immunized with p190-3, a recombinant protein which contains mostly conserved sequences derived from the p190 protein of the K1 parasite isolate. Proliferative T-cell responses of lymph node cells from immunized mice were assessed by stimulation in vitro with p190-3 or preparations of parasitized red blood cells (PRBC) containing the native protein. The p190-3-specific T cells from C57BL/6 mice consistently responded to some P. falciparum isolates, representing either the K1 or MAD20 serotype of p190, but not to other P. falciparum strains or to rodent malaria parasite-infected red blood cells. p190-3-specific T-cell responses from other mouse strains (BALB/c, C3H/He) did not distinguish between P. falciparum isolates. The polymorphic epitopes which were preferentially recognized by T cells from C57BL/6 mice were identified.

We have previously shown that p3-13 (KTIAY-DEEARR) of the 65-kDa heat shock protein (hsp65) of Mycobacterium tuberculosis and Mycobacterium leprae is selected as an important T cell epitope in HLA-DR17+ individuals, by selectively binding to (a pocket in) DR17 molecules, the major subset of the DR3 specificity. We have now further studied the interaction between p3-13, HLA-DR17 and four different TCR (V beta 5.1, V beta 1, and V beta 4) by using T cell stimulation assays, direct peptide-DR binding assays, and a large panel (n = 240) of single amino acid substitution analogs of p3-13. We find that residues 5(I) and 8(D) of p3-13 are important DR17 binding residues, whereas the residues that interact with the TCR vary slightly for each DR17-restricted clone. By using N- and C-terminal truncated derivatives of p2-20 we defined the minimal peptide length for both HLA-DR17 binding and T cell activation: the minimal peptide that bound to DR17 was seven amino acids long whereas the minimal peptide that activated T cell proliferation was eight amino acids in length. Furthermore, two new DR17-restricted epitopes were identified on hsp70 and hsp18 of M. leprae. Alignment of the critical DR17-binding residues 5(I) and 8(D) of p3-13 with these two novel epitopes and two other DR17-binding peptides revealed the presence of highly conserved amino acids at positions n and n + 3 with I, L, and V at position n and D and E at position n + 3. D and E are particularly likely to interact with the DR17-specific, positively charged pocket that we have defined earlier. Based on these results, a set of single amino acid substituted analogs that failed to activate these T cell clones but still bound specifically to DR17 was defined and tested for their ability to inhibit T cell activation by p3-13 or other DR17-restricted epitopes. Those peptides were able to inhibit the response to p3-13 as well as other DR17-restricted mycobacterial epitopes in an allele-specific manner, and are anticipated to be of potential use for immunotherapeutic and vaccine design strategies.

The only peptide of Sendai virus that is recognized by cytotoxic T lymphocytes (CTL) in B6 mice was found with (i) the use of recombinant vaccinia virus constructs containing separate genes of Sendai virus and (ii) a set of overlapping peptides completely spanning the identified nucleoprotein (NP) gene product. This immunodominant NP peptide is recognized by Sendai virus-specific CTL that are known to have therapeutic effects in vivo. By subcutaneous immunization, this peptide induced Sendai virus and NP peptide-specific CTL memory responses in vivo. Most importantly, mice that had been immunized with this peptide were protected against a lethal virus dose, indicating that viral peptides can be used as antiviral T-cell vaccines. The induction of T-cell memory by free peptide immunization potentially has wide applicability in biology and medicine, including protection against infectious disease.

In an effort to serologically differentiate syphilis from yaws, 69 monoclonal antibody species raised against Treponema pallidum subsp. pallidum were tested by immunoblotting for their reactivity with Treponema pallidum subsp. pertenue. All monoclonal antibodies reacted with antigens with the same molecular weight of both subspecies. Furthermore, no differences in reactivity between sera from yaws patients and from syphilis patients were found by Western blot (immunoblot) analysis of cell lysates of T. pallidum subsp. pallidum and T. pallidum subsp. pertenue. We tried to exploit the only known molecular difference between the subspecies. The subunits of the 190-kilodalton multimeric proteins TpF1 and TyF1 of T. pallidum subsp. pallidum and T. pallidum subsp. pertenue, respectively, have previously been shown to differ in one amino acid residue at position 40. In this study, no difference was found in immunoreactivity of TpF1 or TyF1 with either syphilis sera or yaws sera. Synthetic peptides based on the sequence of TpF1 and of TyF1 were used in an enzyme-linked immunosorbent assay with syphilis sera and yaws sera. Again, no difference in reactivity between the T. pallidum subsp. pallidum- and T. pallidum subsp. pertenue-derived peptides was observed.

A panel of murine monoclonal antibodies (MAbs) to the human immunodeficiency virus type 1 trans-activator tat protein were characterized. The anti-tat MAbs were mapped to the different domains of the tat protein by Western blot (immunoblot) and Pepscan analyses. One-half of the MAbs tested mapped to the amino-terminal proline-rich region, and one-third of the MAbs tested mapped to the lysine-arginine-rich region of tat. The individual MAbs were tested for inhibition of tat-mediated trans activation, using a cell-based in vitro assay system. MAbs which mapped to the amino-terminal region of the tat protein demonstrated the highest degree of inhibition, whereas MAbs reactive to other portions of the molecule exhibited a less pronounced effect on tat function.

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 (P0.001) 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 (P0.05). Average daily gain and FE did not differ between E-IM pigs and the surgical castrates, but intact boars performed better than L-IM (P0.02). 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 (P0.03) 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 C(16)-hydrocarbon chain were used. The biostability of the bond between peptide and carrier molecule is thioetheramidedisulfidethioester. However, the immunogenic potency of the constructs used was found to be thioesterdisulfideamidethioether. 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.

STKR is a G protein-coupled receptor that was cloned from the stable fly, Stomoxys calcitrans. Multiple sequence comparisons show that the amino acid sequence of this insect receptor displays several features that are typical for tachykinin (or neurokinin, NK) receptors. Insect tachykinin-related peptides, also referred to as "insectatachykinins," produce dose-dependent calcium responses in Drosophila melanogaster Schneider 2 cells, which are stably transfected with this receptor (S2-STKR). These responses do not depend on the presence of extracellular Ca(2+)-ions. A rapid agonist-induced increase of inositol 1,4,5-trisphosphate (IP(3)) is observed. This indicates that the agonist-induced cytosolic Ca(2+)-rise is caused by a release of Ca(2+) ions from intracellular calcium stores. The pharmacology of STKR is analyzed by studying the effects of the most important antagonists for mammalian NK-receptors on STKR-expressing insect cells. The results show that spantide II, a potent substance P antagonist, is a real antagonist of insectatachykinins on STKR. We have also tested the activity of a variety of natural insectatachykinin analogs by microscopic image analysis of calcium responses in S2-STKR cells. At a concentration of 1 microM, almost all natural analogs produce a significant calcium rise in stable S2-STKR cells. Interestingly, Stc-TK, an insectatachykinin that was recently discovered in the stable fly (S. calcitrans), also proved to be an STKR-agonist. Stc-TK, a potential physiological ligand for STKR, contains an Ala-residue (or A) instead of a highly conserved Gly-residue (or G). Arch.

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.

Small hydrophobic peptides were studied as possible substrates of the multidrug resistance protein (MRP)-1 (ABCC1) transmembrane transporter molecule. As observed earlier for P-glycoprotein- (Pgp; ABCB1) overexpressing cells, MRP1-overexpressing cells, including cells stably transfected with the MRP1 cDNA, showed distinct resistance to the cytotoxic peptide N-acetyl-Leu-Leu-norleucinal (ALLN). Resistance to this peptide and another toxic peptide derivative, which is based on a Thr-His-Thr-Nle-Glu-Gly backbone conjugated to butyl and benzyl groups (4A6), could be reversed by MRP1 inhibitors. The reduced toxicity of 4A6 in MRP1-overexpressing cells was found to be associated with lower accumulation of a fluorescein-labeled derivative of this peptide. Glutathione (GSH) depletion had a clear effect on resistance to ALLN but hardly affected 4A6 resistance. In a limited structure-activity study using peptides that are analogous to 4A6, MRP1-overexpressing cells were found to be resistant to these peptides as well. Remarkably, when selecting A2780 ovarian cancer cells for resistance to ALLN, even in the absence of Pgp blockers, resulting cell lines had up-regulated MRP1, rather than any of the other currently known multidrug resistance transporter molecules including Pgp, MRP2 (ABCC2), MRP3 (ABCC3), MRP5 (ABCCS), and the breast cancer resistance protein ABCG2. ALLN-resistant, MRP1-overexpressing cells were found to be cross-resistant to 4A6 and the classical multidrug resistance drugs doxorubicin, vincristine, and etoposide. This establishes MRP1 as a transporter for small hydrophobic peptides. More extensive structure-activity relationship studies should allow the identification of clinically useful peptide antagonists of MRP1.

Theoretically it seems highly unlikely that relatively small peptides could mimic functionally discontinuous epitopes of antigens. Nevertheless various recent reports show this to be the case. Peptide mimics of protein-, polysaccharide- and DNA-epitopes have been shown to be able to replace the native epitope. Moreover, some of them are able to induce, when used in a vaccine, antibodies with the same activity as that of the antibody used as a template. These mimics, called mimotopes, can be used in vaccines and diagnostics and can be developed more or less systematically using solely antibodies and random, semi-random and dedicated peptide arrays or libraries. Furthermore, the mimotope concept which seems to have proven itself for antibody and antigen interaction can be applied equally well to many receptor ligand interactions and thus may form a new generic approach to the development of drugs. Ltd.

Three rabbit antibodies (R521, R505, R524) were produced, and raised to synthetic peptides corresponding to residues 94-105, 100-111, and 223-234, respectively, of the sheep prion protein (PrP). Epitope mapping analysis revealed the monospecific character of antisera R505 and R524. In addition to the amino acid sequence against which it was raised, R521 also recognized other small epitopes. ELISA and radio-immunoprecipitation were used to assess the relative immunoreactivities of the antisera to the normal sheep prion protein (PrP(c)). Highest reactivity was found for R521, followed by R505 and R524. According to Western blot analysis, all three sera specifically reacted with the prion proteins PrP(Sc) and PrP27-30, extracted from the brain stem of a scrapie-affected sheep. Yet, with R505 not all of the lower molecular weight deglycosylated forms could be detected. Contrary to the immunoreactivities found with the PrP(Sc) and PrP27-30 isoforms, only R521 recognised PrP(c) from a healthy sheep. The usefulness of all three anti-peptide sera in the immunohistochemical detection of PrP(Sc) in brain stem and tonsils of scrapie-affected sheep was demonstrated and compared with an established rabbit anti-PrP serum.

SUMMARY We have previously demonstrated that the Mycobacterium leprae 18-kD heat shock protein (HSP18) is represented among the antigenic targets of human T cell responses induced by M. leprae immunization and that the peptide 38–50 serves as an immunodominant epitope recognized by CD4+ T cell clones. By using peripheral blood mononuclear cells and T cell lines from the same donor group, we have in this study shown that the M. leprae HSP18 and peptide 38–50 were recognized by memory T cells 8 years after immunization with M. leprae. The finding that M. bovis BCG-induced T cell lines responded to M. leprae HSP18, but not to the peptide 38–50, suggested the existence of additional T cell epitopes of a cross-reactive nature. Consistent with this, testing of the T cell lines for proliferative responses to the complete HSP18 molecule, truncated HSP18 (amino acid (aa) residues 38–148) and overlapping synthetic peptides, made it possible to identify two cross-reactive epitope regions defined by aa residues 1–38 and 41–55. While peptide 38–50-reactive T cell clones showed limited cross-reactivity by responding to M. leprae, M. avium and M. scrofulaceum, the T cell lines specific to the epitopes 1–38 and 41–55 were broadly cross-reactive, as demonstrated by their response to M. leprae, M. tuberculosis complex, M. avium and other mycobacteria. MHC restriction analysis of the HSP18-responding T cell lines showed that the epitopes 1–38 and 38–50 were presented by one of the two HLA-DR molecules expressed from self HLA-DRB1 genes, whereas the epitope 41–55 was recognized in the presence of autologous as well as HLA-DR and HLA-DQ mismatched allogeneic antigen-presenting cells. The results obtained in this study made it possible to identify cross-reactive T cell epitopes of the M. leprae HSP18, and provide an explanation for T cell recognition of this antigen in individuals infected with species of the M. tuberculosis complex or environmental mycobacteria.

Vaults are 13 megadalton ribonucleoprotein particles composed largely of the major vault protein (MVP) and two high molecular weight proteins, p240 and p193, and a small vault RNA (vRNA). Increased levels of MVP expression, vault-associated vRNA, and vaults have been linked directly to multidrug resistance (MDR). To further define the putative role of vaults in MDR, we produced monoclonal antibodies against the Mr 193,000 vault protein and studied its expression levels in various multidrug-resistant cell lines. We find that, like MVP, p193 mRNA and protein levels are increased in various multidrug-resistant cell lines. Subcellular fractionation of vault particles revealed that vault-associated p193 levels are increased in multidrug-resistant cells as compared with the parental, drug-sensitive cells. Furthermore, protein analysis of postnuclear supernatants and co-immunoprecipitation studies show that drug-sensitive MVP-transfected tumor cells lack this up-regulation in vault-associated p193. Our observations indicate that vault formation is limited not only by the expression of the MVP but also by the expression or assembly of at least one of the other vault proteins.

By using a synthetic peptide approach, we mapped epitopes from the mycobacterial 65-kDa heat shock protein (HSP65) recognized by human T cells belonging to the Mycobacterium leprae memory repertoire. A panel of HSP65 reactive CD4 + T-cell lines and clones were established from healthy donors 8 years after immunization with heat-killed M. leprae and then tested for proliferative reactivity against overlapping peptides comprising both the M. leprae and Mycobacterium tuberculosis HSP65 sequences. The results showed that the antigen-specific T-cell lines and clones established responded to 12 mycobacterial HSP65 peptides, of which 9 peptides represented epitopes crossreactive between the M. tuberculosis and M. leprae HSP65 (amino acids [aa] 61 to 75, 141 to 155, 151 to 165, 331 to 345, 371 to 385, 411 to 425, 431 to 445, 441 to 455, and 501 to 515) and 3 peptides (aa 343 to 355, 417 to 429, and 522 to 534) represented M. leprae HSP65-specific epitopes. Major histocompatibility complex restriction analysis showed that presentation of 9 of the 12 peptides to T cells were restricted by one of the 2 HLA-DR molecules expressed from self HLA-DRB1 genes, whereas 3 peptides with sequences completely identical between the M. leprae and M. tuberculosis HSP65 were presented to T cells by multiple HLA-DR molecules: peptide (aa 61 to 75) was presented by HLA-DR1, -DR2, and -DR7, peptide (aa 141 to 155) was presented by HLA-DR2, -DR7, and -DR53, whereas both HLA-DR2 and -DR4 (Dw4 and Dw14) were able to present peptide (aa 501 to 515) to T cells. In addition, the T-cell lines responding to these peptides in proliferation assays showed cytotoxic activity against autologous monocytes/macrophages pulsed with the same HSP65 peptides. In conclusion, we demonstrated that promiscuous peptide epitopes from the mycobacterial HSP65 antigen can serve as targets for cytotoxic CD4 + T cells which belong to the human memory T-cell repertoire against M. leprae . The results suggest that such epitopes might be used in the peptide-based design of subunit vaccines against mycobacterial diseases.

Antisera raised against two distinct peptide regions of the Drosophila neurokinin-like receptor NKD were used to immunolocalize tachykinin-receptor-like proteins in the central nervous system of two insect species: the African migratory locust, Locusta migratoria, and the gray fleshfly, Neobellieria bullata. The resulting immunopositive staining patterns were identical for both antisera. Moreover, a very similar distribution of the immunoreactive material was observed in fleshflies and locusts. Immunoreactivity was found in nerve terminals of the retrocerebral complex, suggesting a presynaptic localization of the receptor in this part of the brain. Cell bodies were stained in the subesophageal ganglion: an anterior group of four larger cells and a posterior group of about 20 cells. These cells have axons projecting into the contralateral nervus corporis allati (NCA) II, bypassing the corpus allatum and projecting through the NCA I into the storage part of the corpus cardiacum. In the glandular part of the corpus cardiacum, the glandular adipokinetic hormone-producing cells did not show any immunopositive staining. In the locust, additional immunopositive staining was observed in internolaterally located neurons of the tritocerebrum and in important integrative parts of the neuropil such as the central body and the mushroom bodies.

The murine natural resistance-associated macrophage protein, Nramp1, has multiple pleiotropic effects on macrophage activation and regulates survival of intracellular pathogens including Leishmania, Salmonella and Mycobacterium species. Nramp1 acts as an iron transporter, but precisely how this relates to macrophage activation and/or pathogen survival remains unclear. To gain insight into function, anti-Nramp1 monoclonal and polyclonal antibodies are used here to localise Nramp1 following activation and infection. Confocal microscope analysis in uninfected macrophages demonstrates that both the mutant (infection-susceptible) and wild-type (infection-resistant) forms of the protein localise to the membranes of intracellular vesicular compartments. Gold labelling and electron microscopy defines these compartments more precisely as electron-lucent late endosomal and electron-dense lysosomal compartments, with Nramp1 colocalizing with Lamp1 and cathepsins D and L in both compartments, with macrosialin in late endosomes, and with BSA-5 nm gold in pre-loaded lysosomes. Nramp1 is upregulated with interferon-(gamma) and lipopolysaccaride treatment, coinciding with an increase in labelling in lysosomes relative to late endosomes and apparent dispersion of Nramp1-positive vesicles from a perinuclear location towards the periphery of the cytoplasm along the microtubular network. In both control and activated macrophages, expression of the protein is 3- to 4-fold higher in wild-type compared to mutant macrophages. In Leishmania major-infected macrophages, Nramp1 is observed in the membrane of the pathogen-containing phagosomes, which retain a perinuclear localization in resting macrophages. In Mycobacterium avium-infected resting and activated macrophages, Nramp1-positive vesicles migrated to converge, but not always fuse, with pathogen-containing phagosomes. The Nramp1 protein is thus located where it can have a direct influence on phagosome fusion and the microenvironment of the pathogen, as well as in the more general regulation of endosomal/lysosomal function in macrophages.

The ultimate vaccine is an oral vaccine which given once protects against a multitude of diseases. Furthermore this ultimate vaccine needs to be very stable and inexpensive to produce. Probably this latter condition can be met only if the vaccines are produced in plants. Such vaccines are called 'edible vaccines'. Edible vaccines can be produced in plants in many ways. Using recombinant plantvirus, CPMV, it was shown that plants can produce massive amounts of chimaeric virus particles which protect after a single injection the target animal against disease. The final step, oral administration, is being addressed at present. Preliminary experiments by others suggest that this step may be solved sooner than expected.

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.

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.

Antigenic drift of the major outer membrane protein (MOMP) P2 of nonencapsulated Haemophilus influenzae as observed during persistent infections in patients with chronic bronchitis was mimicked in a rabbit model in which H. influenzae persisted in subcutaneous cages. The antigenic drift resulted from amino acid substitutions in potentially surface-exposed loops of MOMP P2. Since in a rabbit model the appearance of antigenic variants was associated with the presence of strain-specific bactericidal antibodies (L. Vogel, B. Duim, F. Geluk, P. Eijk, H. Jansen, J. Dankert, and L. van Alphen, Infect. Immun. 64:980-986, 1996), we determined the epitope specificities of these bactericidal antibodies. The eight loops of MOMP P2 of H. influenzae d1 were separately expressed as fusion proteins with glutathione S-transferase. Sera of rabbits persistently infected with H. influenzae reacted with the loop 5 and loop 6 fusion proteins in immunoblotting and enzyme-linked immunosorbent assay. For fine mapping of the epitopes with pepscan analysis, overlapping synthetic peptides consisting of 12 amino acids were made. Rabbit sera contained antibodies reacting with peptides derived from loop 5 and peptides containing amino acids of the side of loop 6. In addition, MOMP P2 variant-specific reactions with the amino acids located at the tip of loop 6 were detected. The rabbit sera showed variant-specific complement-dependent bactericidal activities, which were eliminated by affinity chromatography with fusion proteins of loop 6 but not of loop 5. We conclude that, during persistence of H. influenzae in rabbits, variant-specific bactericidal antibodies are elicited to the variable tip of MOMP P2 loop 6.

The mycobacterial 60,000 MW heat shock protein (hsp 60) is a major antigen recognized by mycobacteria-reactive human CD4+ T cells with lymphokine profiles and effector functions consistent with protective immunity. In addition, the presence of a large number of T-cell epitopes presented by several HLA class II molecules makes this antigen relevant to subunit vaccine design. However, the results from animal models as well as human studies suggest that the mycobacterial hsp 60 may induce T-cell-mediated autoimmune conditions. In humans, the expression of HLA-DR4 represents a risk factor for some autoimmune diseases. These observations suggest that the epitopes from the mycobacterial hsp 60 presented to T cells in the context of HLA-DR4 could be relevant to autoimmunity. This is the first report on identification of HLA-DR4-restricted T-cell epitopes from the mycobacterial antigen hsp 60. In total, five epitopes recognized in the context of HLA-DR4 by the M. leprae hsp 60-reactive CD4+ T-cell clones from a subject immunized with M. leprae were defined by synthetic peptides. Two of the epitopes were M. leprae-specific (aa 343-355, aa 522-534), whereas three epitopes were common to M. leprae and M. tuberculosis (aa 331-345, aa 441-455, aa 501-515). However, all of these epitopes belong to the regions that are highly divergent between the mycobacterial hsp 60 and the homologous human hsp 60 sequence, suggesting that the T cells recognizing the mycobacterial hsp 60 in the context of HLA-DR4 may not necessarily induce autoreactivity.

The scrapie-associated form of the prion protein (PrP(Sc)) accumulates in the brain and lymphoid tissues of sheep with scrapie. In order to assess whether detecting PrP(Sc) in lymphoid tissue could he used as a diagnostic test for scrapie, we studied the localization and distribution of PrP(Sc) in various lymphoid tissues collected at necropsy from 55 sheep with clinical scrapie. Samples collected from the spleen, palatine tonsil, ileum, and five different lymph nodes were immunohistochemically stained for PrP(Sc), PrP(Sc) was found to be deposited in a reticular pattern in the center of both primary and secondary lymphoid follicles. In addition, granules of PrP(Sc) were seen in the cytoplasm in macrophages associated with the lymphoid follicles. In 54 (98%) of the 55 scrapie-affected sheep, PrP(Sc) was detected in the spleen, retropharyngeal lymph node, mesenteric lymph node, and the palatine tonsil. However, only in the palatine tonsils was PrP(Sc) present in a consistently high percentage of the lymphoid follicles. PrP was not detected in any of the lymphoid tissues of 12 sheep that had no neurohistopathological signs of a scrapie infection. We conclude that the tonsils are the best-suited lymphoid tissue to be biopsied for the detection of PrP(Sc) in the diagnosis of clinical scrapie in living sheep.

Recently, we developed a panel of monoclonal antibodies (MoAbs) to rat IL-1 beta and found that MoAbs binding to the aminoacid sequences 66-85 and 123-143 of mature rIL-1 beta inhibited the binding of rIL-1 beta to murine EL4 cells. Here we study whether MoAbs to these and other domains of IL-1 interfere with the biological effects of rIL-1 beta in adult male rats in vivo. Administration of rIL-1 beta (1 or 5 micrograms/kg i.v.) enhanced the plasma concentrations of ACTH, corticosterone (CORT) and of IL-6 in a time- (0.5-4 h) and dose-dependent manner. Because 2 h after 5 micrograms/kg i.v., all three parameters were consistently elevated, this dose and time interval was used for further studies. Prior to injection, rIL-1 beta was incubated alone or in the presence of a MoAb (10 mg/kg) for 30 min at 37 degrees C or at 4 degrees C. Plasma ACTH, CORT and IL-6 responses to these mixtures are compared to those obtained after preincubation of rIL-1 beta with a non-IL-1 binding MoAb (PEN7). SILK 3, a MoAb that binds to the 66-85 domain of rIL-1 beta, reduced the ACTH and IL-6 responses by 48 and 45% respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

By combining a DNA subclone and synthetic-peptide approach, we mapped epitopes of the immunogenic mycobacterial 70-kDa heat shock protein (HSP70) recognized by human CD4+ T-cell clones and lines. In addition, we identified the respective HLA-DR molecules used in antigen presentation. The donor groups used were healthy persons immunized with killed Mycobacterium leprae and tuberculoid leprosy patients. The results show that the N-terminal part of the HSP70 molecule contains three different T-cell epitopes, of which two were presented by DR7 (amino acids [aa] 66 to 82 and 210 to 226) and one was presented by DR3 (aa 262 to 274). The C-terminal part contains one epitope (aa 413 to 424) presented by HLA-DR2. The C-terminal epitope shows extensive homology to the corresponding region of the human HSP70 sequence. All of the T-cell epitopes identified were presented by only one particular HLA-DR molecule. We also found that HLA-DR5 and DRw53 can present HSP70 to T cells, demonstrating the presence of additional epitopes not yet defined at the peptide level. On the basis of the donors used in this study, recognition of HSP70 at the epitope level seems to be ruled by the restriction elements expressed by the donor rather than by any difference in reactivity between healthy individuals and patients. In conclusion, mycobacterial HSP70 is relevant to subunit vaccine design since it contains a variety of T-cell epitopes presented in the context of multiple HLA-DR molecules.

Three monoclonal antibodies, LF-hCG-6, -26 and -28, raised against holo-human chorionic gonadotropin and directed against its beta-subunit, have been used to design synthetic peptides with improved affinity. By PEPSCAN analyses with pinbound, overlapping nonapeptides, it was shown that the peptide consisting of amino acids (125-133) of the beta-subunit (i.e., PPSLPSPSR) reacted with monoclonal antibody (LF-hCG-6; the sequence (135-143) (i.e., PGPSDTPIL), with LF-hCG-28; and the C-terminal nonapeptide (137-145) (i.e., PSDTPILPQ), with LF-hCG-26. To determine amino acids essential for binding and those comprising the necessary amino acid core, and to establish the optimal length for binding reactivity, sets of replacement nonapeptides and overlapping octa- to dodecapeptides derived from the beta-subunit sequences (122-134) (i.e., KAPPPSLPSPSRL) and (132-145) (i.e., SRLPGPSDTPILPQ) were synthesized. Amino acid cores were as follows: beta-hCG (125-131) (i.e., PPSLPSP) for monoclonal antibody LF-hCG-6, beta-hCG (135-142) for LF-hCG-28 and beta-hCG (139-145) for LF-hCG-26. Based on the optimal length, parent peptide sequences [beta-hCG (125-133), (137-145) and (135-145)] were synthesized by conventional solid-phase procedures. In addition, based on the results with the replacement peptides, peptide derivatives were produced in which specific single improvements had been combined. The affinities of the monoclonal antibodies for the peptide derivatives, compared to the parent peptide sequences, were at least 700 times better for LF-hCG-6 reactive peptides (6.9 x 10(6) M-1 and10(4) M-1, respectively) and 130 times better for LF-hCG-26 reactive peptides (1.3 x 10(6) M-1 and10(4) M-1, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

CD4 cross-linking by antibodies or its natural ligands triggers a tyrosine kinase activity that is one of the necessary steps in the mechanism of human immunodeficiency virus type 1 (HIV-1)-induced syncytium formation and full Th-cell activation. In this study we mapped a part of the dimerization site of human CD4 to amino acids 87-98 using a bivalent CD4 immunoadhesin and a series of overlapping 12-mer peptides of the D1 domain. The dimerization site we found is part of the complementary determining region (CDR) 3-like region of CD4. Using the three-dimensional structure of other immunoglobulin dimers as a basis, a molecular modeling study was performed to dimerize the D1 domains of CD4. Both the peptide binding studies and molecular modeling studies independently led to the conclusion that the CDR3-like region is part of the CD4 dimerization site. The suggested dimerization of CD4 through its CDR3-like region explains the important role that has been ascribed to this region in Th-cell activation and HIV-1-mediated fusion. Based on this model of the CD4 dimer and published results of different mutational analysis studies, a model was proposed for the complex of the CD4 dimer with two MHC-II molecules. The CD4 dimer allows tight binding to a large surface of MHC-II and the complex of CD4 and MHC-II reconciles mutational analysis studies that were previously incompatible. Moreover, the complex suggests how CD4 can dimerize through ligand binding.

Phospholipase A2 (PLA) is a biochemically fully defined glycoprotein, representing the main allergen of bee venom. We have established CD4+ T cell clones specific to PLA from subjects allergic and nonallergic to bee sting. By screening the epitope specificity of these clones with 62 synthetic overlapping dodecapeptides representing the PLA molecule, two immunogenic epitopes, PLA81-92 and PLA113-124, were identified. Additional screening, using longer peptides of up to 18 residues, revealed a third epitope at position PLA 45-62. These epitopes are recognized by specific T cell clones in association with HLA-DP and -DQ molecules, although HLA-DR-associated responses to PLA exist. Primary in vitro proliferation of unfractionated PBMC from bee sting allergic, hyposensitized, or hyperimmune subjects to PLA-derived peptides revealed the same immunogenic sites as found in the T cell clones. Among the different groups of individuals, proliferative responses to the PLA molecule and fragments thereof were generally higher in allergic patients than in nonallergic subjects. Thus, at least three linear epitopes are involved in T cell recognition of PLA, independently whether or not subjects are allergic.

An immunodominant region on baculovirus-produced parvovirus B19 VP2 capsids was localized between amino acids 259 and 426 by mapping the binding sites of a panel of monoclonal antibodies which recognize determinants on the particles. The binding sites of three monoclonal antibodies were fine-mapped within this antigenic domain. Six VP2-specific monoclonal antibodies recognized determinants common to both the empty capsids and native parvovirus. The defined antigenic region is most probably exposed on the native B19 virion and corresponds to part of the threefold spike on the surface of canine parvovirus particles.

Based on PEPSCAN analyses, a peptide derived from the 25-kDa surface protein of P. falciparum sexual stages (LDTSNPVKT, amino acids 122-130) was recently shown to react with transmission-blocking monoclonal antibodies. Subsequently, a pinbound construct (FDDTDPIKK; six amino acids substituted) was designed that reacted 1000 times better with the transmission-blocking monoclonal antibody 32F81 than with the parent LDTSNPVKT peptide. While this construct was obtained through single amino acid replacement studies, it now is shown that the combined contribution of the various substitutions is necessary to give the total effect. Free peptides comprising LDTSNPVKT or FDDTDPIKK were shown to inhibit the interaction of the transmission-blocking monoclonal antibody with plate-bound peptides, when prepared by conventional synthesis procedures. Compared with peptides that contained LDTSNPVKT, similar levels of inhibition could be achieved with an average 500-fold lower molar amount of peptides containing the FDDTDPIKK sequence. Affinity constants of the peptides for the transmission-blocking monoclonal antibody ranged from 1.3 x 10(5), for peptides that contained LDTSNPVKT, to 2.6 x 10(8), for peptides that contained FDDTDPIKK. The structural relationship between the epitope of the 25-kDa surface protein and the peptides was demonstrated by competition experiments with the transmission-blocking monoclonal antibody 32F81. Again, peptides comprising the newly designed sequence FDDTDPIKK competed about 200 times better than peptides with the parent LDTSNPVKT sequence.

CKS-17 (LQNRRGLDLLFLKEGGL), a synthetic peptide derived from a conserved region of retroviral transmembrane proteins, has previously been shown to suppress several different immune effector mechanisms. The present study was undertaken to further delineate immunosuppressive site(s) of CKS-17. Overlapping hexapeptides covering the complete sequence of CKS-17 were synthesized. One CKS-17-derived hexapeptide, LDLLFL, suppressed ligand [CD3, interleukin (IL)-2]-induced lymphocyte proliferation. Spontaneous proliferation of transformed lymphoid cell lines, as well as cell lines from myeloid or epitheloid origin, was not inhibited by LDLLFL. Full suppression required the continuous presence of LDLLFL during culturing, and did not involve interference with monocyte function. Radiolabeling studies showed that the hexapeptide did not compete with IL-2 for IL-2 receptor binding. Most likely the LDLLFL motif interferes with steps shared by the IL-2 and CD3 receptor-induced signaling pathways. Since LDLLFL displays multiple immunosuppressive activities, it may constitute a biologically relevant immunosuppressive site of retroviral transmembrane proteins.

In many biological processes, defined regions of proteins are involved in selective recognition. These regions can often be mimicked with peptides and are the main targets for vaccine and drug development. The authors review the use of peptides, to define and ultimately mimic defined protein regions of interest. Especially the role of the Pepscan method is emphasized. This method has been proven to be a useful and fast tool in defining protein regions of interest. It is based on the simultaneously synthesis of multiple peptides coupled to solid supports. Hundreds of peptides can be produced and tested in a relatively short period of time. With the construction of random peptide libraries in recombinant DNA systems, it is now even possible to screen for peptidic determinants without the requirement of preliminary knowledge of primary structure. Having this information, the affinity of peptides can be further enhanced with the Pepscan approach. The power of this approach will be illustrated with results from studies on the development of synthetic vaccines and hormone analogues.

Antibody-binding sites were mapped on all overlapping nonapeptides of the major core protein p24 of human immunodeficiency virus type 1 (HIV-1) using murine monoclonal antibodies (MAbs) and sheep and rabbit polyclonal antibodies raised against HIV-1/H9 (strain IIIB) viral lysate and antibodies obtained from humans infected with HIV-1. The binding sites were mapped to various distinct regions of this protein. After superimposition of the antibody-binding sites on a proposed model of p24 of HIV-1, these sites appeared to be located on the surface of the protein on loops, turns and coils of p24 but, unexpectedly, not on the major part of the predicted 'puff'. Little reaction was found with the inaccessible anti-parallel beta-barrel. These results are the first experimental evidence for the validity of the structure proposed for p24 of HIV-1.

The precipitating and neutralizing activities of normal bovine sera with FMDV were studied and compared. Twenty-two out of 79 normal bovine sera gave a positive reaction in micro neutralization tests with FMDV type O, while six did so with type A. In RID tests 32 sera were positive with type O and 28 with type A virus. Almost all of the 79 sera gave a positive reaction in the RID with trypsin treated virus of both types. After three to four fold concentration most sera also gave visible reactions in ID tests when tested against complete virus. When O virus was used the ID patterns produced by most normal sera clearly differed from those obtained with early and late convalescent sera from FMDV infected steers. When type A materials were employed this was also the case but to a lesser extent. The patterns obtained with concentrated normal sera showed, in general a strong line with trypsin treated virus and no line or a weaker one with complete virus. The substances in normal bovine sera precipitating trypsin treated O virus were different from those reacting with trypsinized virus of type A.

Summary Neutralizing monoclonal antibodies raised against intact foot-and-mouth disease virus type O1 reacted with intact virus and trypsin-treated virus particles. Some of the monoclonal antibodies showed a slight but definite reaction with the 12S subunit, but none of them reacted with the isolated capsid protein VP1 or any of the other viral proteins. These results confirm the difference between the neutralizing antigenic determinants exposed on intact virus particle and on isolated capsid protein VP1. They suggest further that the neutralizing antigenic determinant and the cell attachment site are not identical.

Summary Cross-reactions between two strains of foot-and-mouth disease virus (FMDV) belonging to different serotypes (A and O) were studied with intact virus and virus subunits and antisera produced against the isolated structural proteins. Anti-VP1 type O serum showed cross-reactive neutralizing activity, in contrast to the sera raised against intact virus type O, whereas anti-VP1 type A serum only neutralized homologous virus. Anti-VP2, VP3 and VP4 did not show neutralizing activity. In the enzyme-linked immunosorbent assay and radio-immunoassay anti-VP1, -VP2 and -VP3 sera reacted with the 12S virus subunits of both serotypes. No activity was obtained against VP4. Competition experiments with virus subunits of virus type A and O show that anti-VP1 serum is the most type-specific. Anti-VP2 serum is completely cross-reactive, while anti-VP3 serum reacted in an intermediate way. The identical reactions obtained with anti-VP2 sera and the homologous and heterologous virus subunits suggest that the exposed VP2 antigens are identical.

Using polyclonal sera raised against foot-and-mouth disease virus in susceptible animals, evidence was obtained for the existence of at least one further important antigenic site in addition to the neutralizing site on VP1 140–160.