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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Summary Strains of foot-and-mouth disease virus of types O1 and A10 were isolated which showed no significant loss of infectivity upon trypsinization. These ‘trypsin-resistant’ (TR) viruses were obtained by serial passage in BHK cells of virus that was trypsin-treated before inoculation of the cells. Three O1 isolates were cloned and studied further. Cell attachment of those TR O1 variants (OTR1) was not reduced by trypsinization, unlike that of parent virus. The polypeptide compositions of TR viruses as determined by SDS-polyacrylamide gel electrophoresis were identical with those of parent virus, with the exception of OTR1 which contained an additional polypeptide approx. 3000 daltons larger than VP1. After trypsinization, which normally cleaves VP1, the polypeptide composition of the three TR viruses (including OTR1) and of parent virus did not show any significant difference. In OTR1 both the additional virus protein and VP1 were cleaved into a P18 molecule and smaller fragments. The surface location of this additional polypeptide was confirmed by iodination experiments. It was shown by immunodiffusion experiments that only OTR1 differed from the parent virus. This antigenic change was present on the trypsin-sensitive part of the virus since trypsinized TR viruses (including OTR1) were antigenically identical to trypsinized parent virus. The electrophoretic mobilities of the three OTR viruses isolated, and of parent virus, differed somewhat before trypsinization. After trypsin-treatment, the mobilities of TR viruses were all increased to the same level; however, their rate of migration was lower than that of trypsin-treated parent virus. This lower mobility of trypsin-treated OTR viruses was the only difference which could be associated with retained infectivity.

The existence of various molecular weight forms of inhibin in ovarian follicular fluid has been reported earlier, while there is no information on the form of inhibin in testicular tissue. Inhibin bioactivity was therefore estimated in eluates of slices, obtained after SDS-PAGE of rat testicular and ovarian homogenates, rat Sertoli cell-conditioned medium (rSCCM) and bovine ovarian follicular fluid (bFF). The only form of inhibin detected in testes from 22-day-old rats and in rSCCM was a 30 kDa protein. In rat ovarian extracts, larger forms of inhibin were also found as well as the predominant 30 kDa form. An activin-like activity was found in the 25 kDa SDS-PAGE eluates of both rSCCM and ovarian homogenates, which caused a dose-dependent increase of FSH release from cultured pituitary cells. Activin-like activity and several forms of inhibin were found in bFF after SDS-PAGE. After purification of inhibin from bFF using dye affinity, anion-exchange, lentil lectin affinity chromatography and a subsequent reversed phase chromatography step, two pools of inhibin activity were obtained. These were separated by SDS-PAGE revealing 30 and 58 kDa inhibin forms. The immunoactivity of these forms of inhibin was then estimated using antibodies against the 22 N-terminal amino acids of the α subunit of 30 kDa bovine inhibin. It appeared that the two molecular weight forms of inhibin had bioactivity/immunoactivity ratios which differed more than five-fold. This indicates that results of radioimmunoassays of inhibin of ovarian origin, using peptide antisera, should be interpreted with caution. Journal of Endocrinology (1989) 122, 293–301

Antisera were raised against the chemically synthesized peptide corresponding to each epitope of three foot-and-mouth disease virus strains. Peptide synthesis was further used to determine which amino acid residues in each epitope are important for the specificity of antisera raised against the whole virus. The specificity of the antibody paratope for its epitope was shown to depend on structure as well as sequence. Anti-virus sera demonstrated a greater specificity for the homologous peptide than did the anti-peptide sera. Two of the three peptides were able to induce neutralizing antibodies against the homologous virus. The specificities of the antibodies present in the anti-peptide sera were also inferred from the reactions of each with related sets of peptides. The cross-reactions observed for the anti-peptide sera were readily explained in terms of the antibody specificities determined to be present. The findings also suggest that the diversity of antibodies raised against small peptides is limited and is determined by the immune system. A similar limited response to the native protein was observed, which may account for the high frequency with which anti-peptide sera react with the native homologous protein.

Summary Antibody produced against preparations of VP1, one of the four structural polypeptides of foot-and-mouth disease virus, neutralized the virus and reacted with both full and empty particles in radioimmunoassays (RIA). Antiserum against VP2 reacted with artificial empty particles of the virus but not with full particles. In contrast, none of the individual polypeptides of poliovirus produced antisera which neutralized the virus nor reacted with it in RIA. However, antisera produced with VP1 and VP2 reacted with artificial empty particles in RIA.

Summary An enzyme-linked immunosorbent assay for the detection and titration of antibody against Aujeszky's Disease virus in pig sera is described. The sensitivity of the technique compared favourably with that of an indirect immunofluorescence technique and a microtitration virus-neutralization test. Zusammenfassung Ein “Enzyme Linked Immunosorbent Assay” (ELISA) fur den Nachweis von Antikorpern gegenuber dem Virus der Aujuszky-Krankheit in Schweineseren Es wird ein “Enzyme Linked Immunosorbent Assay” (ELISA) fur den Nachweis und die Titerbestimmung von Antikorpern gegenuber dem Virus der Aujjeszky-Krankheit in Schweineseren beschrieben. Die Empfindlichkeit der Technik liegt vergleichsweise hoher als bei der indirekten Immunofluoreszenz und dem Virusneutralisationstest mit der Mikrotechnik.

A procedure is described for rapid concurrent synthesis on solid supports of hundreds of peptides, of sufficient purity to react in an enzyme-linked immunosorbent assay. Interaction of synthesized peptides with antibodies is then easily detected without removing them from the support. In this manner an immunogenic epitope of the immunologically important coat protein of foot-and-mouth disease virus (type O1) is located with a resolution of seven amino acids, corresponding to amino acids 146-152 of that protein. Then, a complete replacement set of peptides in which all 20 amino acids were substituted in turn at every position within the epitope was synthesized, and the particular amino acids conferring specificity for the reaction with antibody were determined. It was found that the leucine residues at positions 148 and 151 were essential for reaction with antisera raised against intact virus. A lesser contribution was derived from the glutamine and alanine residues at positions 149 and 152, respectively. Aside from the practical significance for locating and examining epitopes at high resolution, these findings may lead to better understanding of the basis of antigen-antibody interaction and antibody specificity.

Summary Neutralizing monoclonal antibodies raised against type O foot-and-mouth disease virus have been characterized on the basis of their reactivity with a panel of single site monoclonal antibody-resistant mutants which had defined three antigenic sites. Five antibodies neutralized all these mutants, but by selecting further single site mutants with one of these antibodies it was possible to define a fourth site involved in virus neutralization. Two monoclonal antibodies still neutralized these mutants and all multiple site resistant mutants. One multiple site resistant mutant was resistant to neutralization at each of four antigenic sites but was still efficiently neutralized by type O convalescent cattle sera. The relationship between sites recognized by different monoclonal antibodies generated in different laboratories is discussed.