Mapping and characterization of a sequential epitope on the rabies virus glycoprotein which is recognized by a neutralizing monoclonal antibody, RG719

We have established a murine hybridoma cell line RG719 which produces a rabies virus-neutralizing IgM-type monoclonal antibody (referred to as MAb RG719). Immunoblot analysis indicated that the antibody recognized a sequential epitope of G protein. Among four rabies virus strains tested, the antigenicity to MAb RG719 was absent from the Nishigahara strain, while the other three strains (HEP, ERA and CVS) reacted to the MAb. Studies with deletion mutants of the G protein indicated that the epitope was located in a middle region of the primary structure of G protein, ranging from position 242 to 300. By comparing the estimated amino acid sequence of the four strains, we found in this region two amino acids (at positions 263 and 291) which are common to three of those strains but are not shared by the Nishigahara strain. The site-directed point mutagenesis revealed that replacement of phenylalanine-263 by leucine destroyed the epitope of the HEP G protein, while the epitope was generated on the Nishigahara G protein whose leucine-263 was replaced by phenylalanine. These observations suggest that phenylalanine-263 is essential for constructing the epitope for MAb RG719. The synthetic 20-mer peptide produced by mimicking the amino acid sequence (ranging from amino acid positions 249 to 268) of the presumed epitope region was shown to bind specifically to MAb RG719 and also to raise the virus-neutralizing antibodies in rabbits. Vaccination with the HEP vaccine produced in Japan induced in humans and rabbits production of significant amounts of the antibodies which reacted with the 20-mer peptide.     

Unstable expression and thermal instability of a species-specific cell surface epitope associated with a 66-kilodalton antigen recognized by monoclonal antibody EM-7G1 within serotypes of Listeria monocytogenes grown in nonselective and selective broths

Conditions that resulted in unstable expression and heat instability of a cell surface epitope associated with a 66-kDa antigen in Listeria monocytogenes serotypes were identified with the probe monoclonal antibody (MAb) EM-7G1 in an enzyme-linked immunosorbent assay. This epitope appeared to be absent in three serotypes (serotypes 3b, 4a, and 4c), which did not react with MAb EM-7G1 irrespective of the enrichment broth tested. The remaining 10 serotypes were detected by MAb EM-7G1 only when cells were grown in nonselective brain heart infusion broth (BHI) or selective Listeria enrichment broth (LEB). When cells were grown in Listeria repair broth (LRB), only 6 of the 13 serotypes were detected by MAb EM-7G1, and recognition of serogroup 4 was completely lost. None of the 13 serotypes was detected by MAb EM-7G1 when cells were grown in two other commonly used Listeria-selective media, UVM1 broth and Fraser broth (FRB), indicating that possible loss of epitope expression occurred under these conditions. MAb EM-7G1 maintained species specificity without cross-reacting with live or heat-killed cells of six other Listeria spp. (Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria grayi, and Listeria murrayi) irrespective of the enrichment conditions tested. Due to heat instability of the cell surface epitope when it was exposed to 80 or 100 degrees C for 20 min, MAb EM-7G1 is suitable for detection of live cells of L. monocytogenes in BHI or LEB but not in LRB, UVM1, or FRB enrichment medium.     

Radiotherapy in the treatment of dermatologic malignancies

A large set of monoclonal antibodies (MAbs) directed against the fusion glycoprotein complex F1F2 of bovine respiratory syncytial virus (BRSV) and several polyclonal sera from infected or vaccinated animals were tested in Pepscan to locate linear epitopes on the F-protein. The polyclonal sera mapped to antigenic sites that correspond exactly to known antigenic sites on the F protein of human RSV. Only the neutralizing MAb 3 could be mapped with Pepscan. MAb 3 reacted with three successive overlapping linear peptides that shared the amino acid sequence 173STNKAVVSLS182. The sequence of this novel neutralization site is conserved in all known BRSV- and human RSV-strains and is located on the N-terminus of F1, adjacent to the hydrophobic, putative fusion-related region. This region is probably part of a central coiled-coil stem that is structurally conserved in paramyxovirus fusion and orthomyxovirus hemagglutinin glycoproteins. This linear conserved epitope may be a potential candidate for a peptide-based vaccine which can induce neutralizing antibodies against all groups and subgroups of RSV. Furthermore, the proposed structural features of the neutralization site may aid in the design of a peptide-based vaccine.     

Two dominant neutralizing antigenic determinants of canine parvovirus are found on the threefold spike of the virus capsid

The 25-nm diameter parvovirus capsid is assembled from 60 copies of a sequence common to the overlapping VP1 and VP2 proteins. Here we examine the epitope specificity's of 28 monoclonal antibodies (MAb) prepared against canine parvovirus (CPV), feline panleukopenia virus (FPV), and raccoon-dog parvovirus or blue (Arctic) fox parvovirus. Comparing the reactivity of those MAb with various MAb-selected escape mutants, or with natural variants of CPV or mink enteritis virus (MEV) which differ at known sequences, showed that the binding of 20 of those MAb was strongly affected by variations of two regions on the threefold spike of the CPV capsid. One region was adjacent to the tip of the threefold spike, and the second was around VP2 residue 300, on the shoulder of that structure. MAb recognizing both antigenic sites efficiently neutralized the virus infectivity and inhibited hemagglutination. Mutations leading to natural antigenic variation have also been observed in both those sites in naturally variant strains of CPV or MEV, suggesting that they are important antigenic structures on these parvoviruses. The bindings of several MAb were not affected by the mutations at those antigenic sites, indicating that they recognized other, and perhaps conserved, structures.     

Abnormal sex development and impotence

OBJECTIVE: Cross-reactivity of anti-Sm autoantibodies with a certain ribosomal protein has been reported previously. The present study was undertaken to identify the anti-Sm-reactive ribosomal protein, and to characterize the cross-reactive epitope. METHODS: Two-dimensional gel electrophoresis followed by immunoblotting was used to identify the ribosomal protein (S10) which was reactive with the Y12 anti-Sm monoclonal antibody (MAb). Human anti-Sm antibodies were also tested for cross-reactivity with the Sm-B/B', Sm-D, and isolated S10 proteins by immunoblotting. Epitope analysis was performed by immunoprecipitation of in vitro-translated products of the recombinant S10 and its various mutants. RESULTS: The Y12 MAb and the affinity-purified human anti-Sm autoantibodies cross-reacted with ribosomal S10 protein. Reactivity of the Y12 MAb with S10 protein was abolished by deletion of 19 amino acids at the carboxyl-terminus of S10, containing the Gly-Arg-Gly sequence motif shared by Sm-B/B' and Sm-D (D1 and D3). Replacements of Arg-158 with Gly and of Arg-158/Arg-160 with Gly/Gly at the carboxyl-terminal 157-Gly-Arg-Gly-Arg-Gly region disrupted the Y12 MAb recognition. CONCLUSION: At least a part of human anti-Sm antibodies and Y12 MAb show cross-reactivity among Sm-B/B', Sm-D, and ribosomal protein S10. The carboxyl-terminal Gly-Arg-Gly region of S10 protein is involved in constructing the cross-reactive epitope. This demonstrates that a common structural feature is shared by the ribosomal protein and the small nuclear RNP proteins.     

A neutralizing monoclonal antibody previously mapped exclusively on human immunodeficiency virus type 1 gp41 recognizes an epitope in p17 sharing the core sequence IEEE

We report here that a human immunodeficiency virus type 1 (HIV-1)-specific neutralizing monoclonal antibody (MAb 1575) mapped to the conserved putative intracellular region from amino acid residues 735 to 752 (735-752 region) of gp41 also recognizes a region in an extracellular portion of p17. Both epitopes have a core recognition sequence (IEEE) in a nonhomologous context. The IEEE motif found in HIV-1 p17 is located in a region known as HGP-30 (residues 86 to 115) which has been previously associated with virus neutralization, cytotoxic T lymphocyte activity, and mother-to-child transmission. An analysis of available gp41 and p17 sequences demonstrates that in these regions both IEEE sequences are highly conserved in different HIV-1 clades. The presence of the IEEE epitope in p17 allows us to explain some unexpected neutralizing characteristics of MAb 1575. In addition, the gp41 735-752 region has been previously reported both in intra- and extracellular locations. Our results suggest that the extracellular location was the result of cross-reactivity with p17.     

Comparative analysis of class I, II and III epitope-detecting CD34 monoclonal antibodies by quantitative flow cytometry

The aim of this study was to compare different CD34 monoclonal antibodies (MAbs) belonging to three different classes: MY10 class I, QBend10 class II, a mixture of three selected MAbs class I and II designated as CD34 Pool, and 8G12 class III. Bone marrow (BM) samples from 13 healthy donors were analyzed for: 1) percentage of CD34+ cells, 2) quantitative expression of CD34 epitopes (antigen's density - AgD) using a quantitative indirect immunofluorescence (QIFI) test, 3) study of CD34+ cell subsets defined by CD34 and CD38 coexpression. 8G12 MAb showed the highest reactivity with regard to the percentage of detected CD34+ cells and AgD on these cells. A nearly identical percentage of CD34+ cells was detected with CD34 Pool, but with a lower AgD. With QBend10, the percentage of CD34 expressing cells was insignificantly decreased and the AgD was slightly lower. The expression of the MY10 epitope was the lowest and was detected on the lowest number of CD34+ cells. Concerning CD34 and CD38 coexpressing subset, we observed that 8G12 class III MAb detected CD34loCD38dim cells with comparable efficiency with MY10 class I MAb, but with significantly higher level than QBend10 class II and CD34 Pool class I+II MAbs. The CD34hiCD38dim subset was detected with the same efficiency by QBend10, CD34 Pool or 8G12 MAbs but with significantly higher frequency than MY10 MAb. In conclusion: class II and III MAbs appear preferable for flow cytometric quantification of CD34+ cells; for CD34+ cell subsets determination class III MAbs should be suitable.     

Basic fibroblast growth factor prevents cAMP-induced apoptosis in cultured Schwann cells

The periplasmic-flagellum (PF) proteins of Triton X-100-soluble and Triton X-100-insoluble sodium dodecyl sulfate-treated fractions from reference and field strains of Serpulina hyodysenteriae, Serpulina innocens, and Serpulina pilosicoli were characterized by Western blotting with a rabbit polyclonal antibody (PAb) specific for the 44-kDa PF sheath protein of S. hyodysenteriae (Z. Li, F. Dumas, D. Dubreuil, and M. Jacques, J. Bacteriol. 175:8000-8007, 1993) and a murine monoclonal antibody (MAb), designated 7G2, specific for the PF core FlaB proteins of S. hyodysenteriae. The MAb 7G2 reacted with a conserved epitope present in the 37-, 34-, and 32-kDa PF core FlaB proteins of all Serpulina species. This suggested that the core FlaB proteins are conserved among porcine Serpulina species. An immunoreactive band of approximately 44 kDa was present with all S. hyodysenteriae, S. innocens, and S. pilosicoli strains that were reacted with the PAb. The specificities of the PAb and the MAb for the FlaA1 and FlaB proteins of Serpulina species were confirmed by N-terminal amino acid sequencing of 44- and 37-kDa proteins, respectively, of S. hyodysenteriae and S. pilosicoli. Results from this study provide further evidence that the 44-kDa protein FlaA1 and the 37-, 34-, and 32-kDa FlaB proteins are conserved among porcine Serpulina species.     

Double-blind, placebo-controlled study of ramipril in diabetics with mild to moderate hypertension

The capacity of a human monoclonal antibody (MAb 33G2) to interfere in vitro both with Plasmodium falciparum merozoite invasion and cytoadherence of infected erythrocytes to melanoma cells has been reported. MAb 33G2 cross-reacts with several P. falciparum antigens but shows highest reactivity with repeated sequences in the asexual blood stage antigen Pf332. This study was conducted in order to further analyze the cytoadherence inhibition mediated by MAb 33G2 and to evaluate the relative contribution of antibodies to Pf332 in the inhibitory activity of immunoglobulins from P. falciparum immune donors. We show here that MAb 33G2 inhibits cytoadherence of infected erythrocytes (PRBCs) with similar efficiency independently of the strain of parasite, while the inhibitory capacity of immunoglobulin fractions from Liberian immune donors was restricted to some strains only. There appears to be no correlation between the reactivity with Pf332 of immunoglobulin preparations from different donors and their capacity to inhibit cytoadherence of PRBCs to melanoma cells. In contrast to MAb 33G2, polyclonal antibodies affinity purified on the Pf332 peptide containing the epitope seen by the MAb showed little or no inhibition of cytoadherence of infected erythrocytes.     

Comparison of neutralizing epitopes among infectious bursal disease viruses using radioimmunoprecipitation

Monoclonal antibodies (MAbs) were used to analyze the relatedness of neutralizing epitopes of infectious bursal disease virus (IBDV) strains. The MAb B69 neutralized the homologous D78 virus but not the MD and Del-A variant strains of IBDV. MAbs 33E8 and R63 neutralized D78 and variant strains MD and Del-A. A cDNA clone consisting of a 1000-base-pair fragment of the VP2 gene from the Del-A IBDV strain was translated using an in vitro system. Six peptides were observed following translation, which represented a full-length product (35.5 kilodaltons) and five truncated products. The translated peptides were radioimmunoprecipitated using MAbs B69, 33E8, and R63. Only MAb R63 immunoprecipitated the in vitro translation products from Del-A. MAbs B69 and 33E8 did not immunoprecipitate the in vitro-translated VP2 peptides. The D78 and Del-A viruses were radiolabeled in vivo using 35S-methionine. Proteins from these viruses were examined by radioimmuno-precipitation with MAbs B69, 33E8, and R63. Although the background made interpretation of the results difficult for MAb B69, this MAb clearly immunoprecipitated VP2 of D78. MAb 33E8 immunoprecipitated the D78 VP3 protein, and R63 immunoprecipitated the VP2 protein. MAb R63 also immunoprecipitated the 35S-methionine-labeled VP2 protein from Del-A. It was concluded that the neutralizing epitope represented by 33E8 was located on VP3 and that the epitope represented by R63 is located on both classic and variant viruses in the region of VP2 that is generally thought to contain sequence variability among IBDV strains.     

Mapping of a protective epitope of the CopB outer membrane protein of Moraxella catarrhalis

A monoclonal antibody (MAb) (MAb 10F3) directed against the CopB outer membrane protein of Moraxella catarrhalis previously was found to enhance pulmonary clearance of M. catarrhalis in an animal model (M. Helminen, I. Maciver, J. L. Latimer, L. D. Cope, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 61:2003-2010, 1993). In the present study, this same MAb was shown to exert complement-dependent bactericidal activity against this pathogen in vitro. Nucleotide sequence analysis of the copB gene from two MAb 10F3-reactive and two MAb 10F3-unreactive strains of M. catarrhalis revealed that the deduced amino acid sequences of these four CopB proteins were at least 90% identical. Comparison of the amino acid sequences of these proteins allowed localization of possible MAb 10F3 binding sites to five relatively small regions of the CopB protein from M. catarrhalis O35E. When five synthetic peptides representing these regions were tested for their ability to bind MAb 10F3 in a direct enzyme-linked immunosorbent assay system, an oligopeptide containing 26 amino acids was shown to bind this MAb. The actual binding region for MAb 10F3 was localized further through the use of overlapping decapeptides that spanned this 26-mer. A fusion protein containing the same 26-mer readily bound MAb 10F3 and was used to immunize mice. The resultant antiserum contained antibodies that reacted with the CopB protein of the homologous M. catarrhalis strain in Western blot analysis and bound to the surface of both homologous and heterologous strains of M. catarrhalis.     

Detailed characterization of the binding site of the lipoprotein lipase-specific monoclonal antibody 5D2

Monoclonal antibody (MAb) 5D2 recognizes lipoprotein lipases (LPL) from different species but not related lipases. This MAb is a unique reagent, used world-wide, because it differentiates between monomeric inactive and dimeric active LPL, inhibits human LPL enzyme activity, and binds to C-terminal LPL sequences involved in interactions with lipoproteins, lipoprotein receptors, and heparin. In this study we have analyzed the fine specificity of the MAb epitope recognition in order to better understand its functional properties and species-specific LPL immune reactivity. In peptide scan assays, MAb 5D2 reacted with all, except two, 13 amino acid-long peptides located between positions 380 and 410. Peptides from the amino terminal end of this region reacted more strongly than those from the carboxyl terminal end. Furthermore, only a peptide from the amino terminal end competed effectively with the binding of MAb 5D2 to native LPL bound to microtiter plates or nitrocellulose. A systematic peptide mutagenesis study indicated that 8 amino acids of the reactive region, mainly located in the amino terminal end, are critical for binding and probably directly interact with MAb 5D2. The experimentally determined antigenicities of species-specific LPL peptides and of the corresponding denatured full-length LPL proteins on immunoblots were consistent with these findings. According to a proposed 3D-model for LPL, only the amino terminal end of the antigenic region is easily surface-accessible. These data combined with 3D-modelling of monoclonal antibody (MAb)-lipoprotein lipase (LPL) protein interaction provide new insight into the known biological effects of MAb 5D2 on LPL and the antigenic determinants that are recognized.     

Differential distribution of the HNK-1 carbohydrate epitope in the vertebrate retina

The expression of the cell adhesion-related HNK-1 carbohydrate epitope in the retina and ciliary body was studied in different vertebrates and in man. A series of eyes from 4 fish, 5 bird, and 9 mammalian species was analyzed by immunohistochemistry with monoclonal antibodies (MAb) HNK-1 and VC1.1 to the HNK-1 epitope, and with MAb SY38 to synaptophysin. Additionally, 7 morphologically normal human eyes were studied. In all fishes, as well as in baboons and man, the radial glia and all retinal layers except the photoreceptor cell layer were immunoreactive for the HNK-1 epitope. In all birds, the nerve fiber layer and both plexiform layers were labelled. In nonprimate mammals only the plexiform layers were immunoreactive. Fine differences in this general immunoreaction pattern were seen in different species. Mab SY38 labeled both plexiform layers of mammals only. In the ciliary body, immunoreaction for the HNK-1 epitope was seen in the inner connective tissue layer only in man, but the ciliary nerves were labelled in all species except the mouse and rat. The HNK-1 epitope seems to be phylogenetically conserved in the retina, where the HNK-1 immunoreactive plexiform layers possibly are overlapped with HNK-1 reactive radial glial cells in fishes and primates. Instead in the inner connective tissue layer of the ciliary body, the HNK-1 epitope is not phylogenetically conserved.     

Structure of a neutralizing antibody bound monovalently to human rhinovirus 2

The structure of a complex between human rhinovirus 2 (HRV2) and the Fab fragment of neutralizing monoclonal antibody (MAb) 3B10 has been determined to 25-A resolution by cryoelectron microscopy and three-dimensional reconstruction techniques. The footprint of 3B10 on HRV2 is very similar to that of neutralizing MAb 8F5, which binds bivalently across the icosahedral twofold axis. However, the 3B10 Fab fragment (Fab-3B10) is bound in an orientation, inclined at approximately 45 degrees to the surface of the virus capsid, which is compatible only with monovalent binding of the antibody. The canyon around the fivefold axis is not directly obstructed by the bound Fab. The X-ray structures of a closely related HRV (HRV1A) and a Fab fragment were fitted to the density maps of the HRV2-Fab-3B10 complex obtained by cryoelectron microscope techniques. The footprint of 3B10 on the viral surface is largely on VP2 but also covers the VP3 loop centered on residue 3064 and the VP1 loop centered on residue 1267. MAb 3B10 can interact directly with VP2 residue 2164, the site of an escape mutation on VP2, and with VP1 residues 1264 to 1267, the site of a deletion escape mutation. Deletion of these residues shortens the VP1 loop, moving it away from the MAb binding site. All structural and biochemical evidence indicates that MAb 3B10 binds to a conformation epitope on HRV2.     

Antigenic structure of the central conserved region of protein G of bovine respiratory syncytial virus

Epitopes were resolved at the amino acid level for nine monoclonal antibodies (MAbs) directed against the central conserved region of protein G of bovine respiratory syncytial virus (BRSV-G). Peptide binding studies showed which amino acids in the epitope contributed to antibody binding. The details of the epitopes were compared with the high-resolution structure of a synthetic peptide corresponding to the central conserved region of BRSV-G, and this indicated which face of the central conserved region is the antigenic structure. The major linear epitope of the central conserved region of BRSV-G is located at the tip of the loop, overlapping a relatively flat surface formed by a double disulfide-bonded cystine noose. At least one, but possibly two sulfur atoms of a disulfide bridge that line the conserved pocket at the center of the flat surface, is a major contributor to antibody binding. Some of the residue positions in the epitope have mutated during the evolution of RSV-G, which suggests that the virus escaped antibody recognition with these mutations. Mutations that occur at positions 177 and 180 may have only a local effect on the antigenic surface, without influencing the structure of the backbone, whereas mutations at positions 183 and 184 will probably have major structural consequences. The study explains the antigenic, structural, and functional importance of each residue in the cystine noose which provides information for peptide vaccine design. Additionally, analysis of the epitopes demonstrated that two point mutations at positions 180 and 205 define the preliminary classification of BRSV subgroups.     

Preparation of an activity-inhibiting monoclonal antibody against human placental aromatase cytochrome P450

We produced a murine monoclonal antibody (MAb) to human placental aromatase cytochrome P450. This MAb, designated MAb3-2C2, was selected on its ability to suppress aromatase activity. The specificity of this MAb was assessed by selective immunoprecipitation of 125I-labeled aromatase cytochrome P450 as well as by the identification of a 55-kDa protein, which was enriched and purified by immunoaffinity chromatography on a MAb-coupled Sepharose 4B column. The MAb was able to suppress both human placental and ovarian microsomal aromatase. Species differences of aromatase were recognized by MAb3-2C2 on the basis of differential immunosuppression of aromatase activity. The antibody had no effect on non-aromatase cytochrome P450s. MAb3-2C2 gave negative results with human placental aromatase P450 in the Western blot analysis. The data presented indicate that MAb3-2C2 is specific for aromatase cytochrome P450 and that its epitope is located in a fragile tertiary conformation of the enzyme, thus making it capable of sensitively affecting catalysis.     

Preferential formation and decreased removal of cisplatin-DNA adducts in Chinese hamster ovary cell mitochondrial DNA as compared to nuclear DNA

We report here evaluation of a competitive enzyme-linked immunosorbent assay (c-ELISA) for detection of Salmonella spp. in chicken organs and faeces. The c-ELISA used a monoclonal antibody (MAb), specific for a genus-specific epitope of the outer core oligosaccharide of salmonellae. Salmonella lipopolysaccharide (LPS) in samples competed with Salmonella LPS coated on microtitre plates, for binding to the MAb. Competition reduced binding of the MAb to the LPS on the plate and of the secondary antibody to the MAb hence reducing the chromogenic signal. Stable coating and minimal false positive were achieved by conjugating LPS to poly-L-lysine. The c-ELISA was compared with motility enrichment culture using modified semisolid Rappaport Vassiliadis (MSRV) medium, which detected less than 10(2) CFU/g, and did not allow migration of non-salmonella species. The c-ELISA detected 10(6) CFU of enriched culture or 10(2)-10(3) CFU of Salmonella/g of faeces. Its limit of detection was thus higher than that of MSRV culture and it had a sensitivity of 92.9% and a specificity of 96.7%.     

Conformational dependence of Anaplasma marginale major surface protein 5 surface-exposed B-cell epitopes

The Anaplasma marginale outer membrane is composed of immunogenic major surface proteins (MSPs) linked both covalently and noncovalently in multimeric complexes (M. C. Vidotto, T. C. McGuire, T. F. McElwain, G. H. Palmer, and D. P. Knowles, Infect. Immun. 62:2940-2946). Consequently, effective induction of antibody against surface-exposed MSP epitopes has been postulated to require maintenance of MSP secondary through quatenary structures. Using MSP5 as a model and the approach of epitope mapping with recombinant expressed full-length and truncated proteins, we demonstrated that the immunodominant surface epitope bound by monoclonal antibody (MAb) ANAF16C1 required disparate amino- and carboxy-terminal regions of MSP5, indicating the conformational dependence of this epitope. The required amino-terminal MSP5 region included the cysteines involved in intramolecular disulfide bonding. The dependence of the immunodominant epitope on disulfide bonding was confirmed by loss of MAb ANAF16C1 binding to MSP5 following disulfide bond reduction and covalent modification of the reduced sulfhydryl groups. The recognition of the MSP5 immunodominant epitope by antibody induced by protective immunization with A. marginale outer membranes was also conformationally dependent, as shown by the loss of epitope binding following serum adsorption with native but not reduced and denatured A. marginale. Importantly, the antibody response to all immunodominant MSP5 surface epitopes was restricted to conformationally dependent epitopes, since the binding of polyclonal anti-MSP5 antibody to the A. marginale surface could be blocked by adsorption with native but not denatured and reduced MSP5. These results confirm the importance of the secondary and tertiary structures of MSP epitopes as immune system targets and support the testing of immunogens which maintain the required conformation.     

Induction of a T- and B-cell response against a unique amino acid sequence of the mouse IgG2A hinge region in a MAb-treated patient

A patient with malignant glioblastoma was treated with intratumoral infusions of the murine MAb425 (IgG2A) directed against the epidermal growth factor receptor. At the 10th infusion, the patient developed somnolence, fever and headache. The symptoms increased during the subsequent 48 hr but then gradually disappeared within a week. The cerebrospinal fluid (CSF) contained increased concentrations of interleukin-2. The main CSF cell subset was CD4 T-cells. A marked blood lymphocyte proliferative response against mouse IgG2A was noted. The reactive T-cell epitope(s) could be localized to a 14 amino acid (RGPTIKPCPPCKCP) long peptide of the hinge region. A B-cell response (IgG antibodies) against this peptide was also induced.     

Mapping the minimal murine T cell and B cell epitopes within a peptide vaccine candidate from the conserved region of the M protein of group A streptococcus

The highly conserved C-terminus of the M protein of group A streptococcus (GAS) is a promising vaccine candidate. An epitope within the conserved C-terminus of the M protein, peptide 145 (a 20-mer with the sequence: LRRDLDASREAKKQVEKALE), has been defined which is the target of opsonic antibodies in both humans and mice, and is recognized by the sera of most adults living in areas of high streptococcal exposure. However, due to potential cross-reactivity between T cells stimulated by this region of the M protein and host cardiac myosin, it is critical to define precisely the minimal protective epitopes within p145. Studies have shown that the immunodominant epitope expressed by p145 is conformational, occurring as an alpha-helical coiled-coil. To enable us to map the murine minimal B cell and T cell epitopes within p145, we have used a novel strategy that allowed us to present shorter sequences of p145 in a native-like conformation. The minimal B cell epitope was found to be contained within residues 7-20 of the p145 sequence, and we have shown that mice immunized with this region are able to generate antibodies that bind to and also opsonize the organism GAS. The T cell epitope is located at the N-terminal region of the p145 sequence, residues 3-14. We have managed, therefore, to define a vaccine candidate--a minimal opsonic B cell epitope within the p145 sequence--that does not incorporate a potentially deleterious T cell epitope.