Can active immunization redirect an anti-IgE immune response?

We used as a template a mouse monoclonal antibody against IgE to isolate peptides from random peptide phage display libraries. Thereby, two types of peptides were isolated that corresponded to two different epitopes on the human IgE molecule. These peptides, also called mimotopes, seem to be a suitable tool in conjunction with carriers to induce an autoimmune response with a beneficial effect in humans, because the originally used template antibody is capable of neutralizing IgE, is nonanaphylactogenic, and inhibits IgE synthesis. The vaccination approach is further supported by the fact that we were capable of isolating anti-idiotypic antibodies from antibody phage display libraries against the template antibody. These anti-idiotypic antibodies were inhibited by both of the isolated IgE mimotopes. Thus, active vaccination with defined IgE mimotopes may represent a follow-up drug for the presently used anti-IgE antibodies.     

An improved enzyme linked immunosorbent assay for detection of anti-ranavirus antibodies in the serum of the giant toad (Bufo marinus)

An improved ranavirus antibody ELISA (R Ab ELISA) for the specific detection of anti-ranavirus antibodies in toad sera was developed. Sheep anti-epizootic haematopoietic necrosis virus (EHNV) was used as the antigen-capture antibody. EHNV was used as the antigen and sera from field and challenged toads were used to detect the virus. Rabbit anti-toad IgG and IgM were used to detect bound toad antibody. Pre-absorption of toad sera with a monoclonal antibody, raised against the 50 kDa EHNV protein, improved the specificity of the technique. A blocking ELISA, immunofluorescence and immuno-electron microscopy were used to confirm the validity of the ELISA. The assay has potential use in screening sera from Bufo marinus for the presence of antibodies against ranaviruses and to facilitate understanding of the humoral immunological response in toads during virus infection.     

Eradication of residual disease by administration of leukemia-specific T cells after experimental allogeneic bone marrow transplantation

Phage display selection strategies rely on the physical link between the displayed heterologous protein ligand and the DNA encoding it. Thus, genes expressing a ligand with a specific binding affinity can be selected rapidly. To improve the specificity and sensitivity of this technology for potential use in identifying ligands to a specific antibody present in a complex mixture, we incorporated a DNA selection step along with the phage display technology. Ligands for hepatitis C virus (HCV) antibodies present in serum were identified by panning a phage-displayed random peptide library against pools of serum HCV antibodies. An additional DNA hybridization screening step using single-stranded DNA isolated from one of the pools increased the specificity and sensitivity, resulting in the selection of an HCV antibody ligand with diagnostic potential.     

Mutagenicity and amount of chloroform after chlorination of bank filtered lake water

A one-step immunoassay for simultaneous detection of serum IgG and IgM antibodies to Borrelia burgdorferi has been developed. The assay is based on C1q, which binds to immune complexes containing IgG and/or IgM antibodies. Micro-beads pre-coated with antibodies to human C1q are mixed with human serum samples and fluorochrome-labelled B. burgdorferi flagellum antigen. In the presence of serum IgG and/or IgM antibodies to B. burgdorferi, fluorochrome-labelled antigen/antibody complexes are formed. These are then bound by serum C1q and are subsequently captured on the anti-C1q-coated beads. The sample is analysed on a flow cytometer and the presence of fluorescent beads is, thus, indicative of a positive test result. In the present study the sensitivity and specificity of the assay are compared to those of the indirect IDEIA B. burgdorferi IgG and the mu-chain capture IDEIA B. burgdorferi IgM ELISAs for separate determination of IgG and IgM. Detection using a flow cytometer can be performed without separation of the beads from the reaction mixture, which means that in practice, the method is carried out as a one-step assay and it is, thus, very suitable for automation. Other advantages of this kind of assay includes an antibody/antigen reaction which occurs in solution and the potential of using the method for the detection of antibodies against several antigens from the same or different infectious agents (multi-parameter screening).     

Phage mimotopes of monoclonal antibodies against plasma membrane Ca2+-ATPase

Libraries of random phage-displayed pentadeca- and hexapeptides were screened with the use of four monoclonal antibodies against the human plasma membrane Ca2(+)-ATPase. Bacteriophages specifically binding the antibodies were selected, and the amino acid sequences of the expressed peptides (mimotopes) were determined. Mimotopes for three antibodies (8B8, 2D8, F9) did not correspond to the Ca2(+)-ATPase sequence. Pentadecapeptides for the 7C8 antibodies displayed similarity to the fragment Glu1097-Arg1113 of the Ca2(+)-ATPase calmodulin-binding site. However, these antibodies failed to bind recombinant fragment Leu1069-Leu1220; therefore, the structure of this epitope remains obscure. This work opens a series of studies of the plasma membrane Ca2(+)-ATPase structure by means of monoclonal antibodies and the phage display method.     

Towards a solution for hepatitis C virus hypervariability: mimotopes of the hypervariable region 1 can induce antibodies cross-reacting with a large number of viral variants

The hypervariable region 1 (HVR1) of the putative envelope protein E2 of hepatitis C virus (HCV) is the most variable antigenic fragment in the whole viral genome and is mainly responsible for the large inter-and intra-individual heterogeneity of the infecting virus. It contains a principal neutralization epitope and has been proposed as the major player in the mechanism of escape from host immune response. Since anti-HVR1 antibodies are the only species shown to possess protective activity up to date, developing an effective prevention therapy is a very difficult task. We have approached the problem of HVR1 variability by deriving a consensus profile from >200 HVR1 sequences from different viral isolates and used it as a template to generate a vast repertoire of synthetic HVR1 surrogates displayed on M13 bacteriophage. This library was affinity selected using many different sera from infected patients. Phages were identified which react very frequently with patients' sera and bind serum antibodies that cross-react with a large panel of HVR1 peptides derived from natural HCV variants. When injected into experimental animals, the 'mimotopes' with the highest cross-reactivity induced antibodies which recognized the same panel of natural HVR1 variants. In these mimotopes we identified a sequence pattern responsible for the observed cross-reactivity. These data may hold the key for future development of a prophylactic vaccine against HCV.     

The effects of interferon-gamma on the central nervous system

A 165bp DNA fragment derived from the 12 kDa subunit of Echinococcus granulosus antigen B (AgB), a major hydatid cyst fluid antigen was cloned in the pMa1-c2 expression vector. A 52 kDa maltose binding-AgB fusion protein (rAgB.MBP) was produced and inclusion bodies containing the fusion protein were solubilised in urea and affinity purified on an amylose-Sepharose 6B column. The immunogenicity of the purified recombinant antigen for IgG4 antibody detection was tested with human serum using immunoblotting, ELISA and dot-ELISA assays and compared to native AgB. Both recombinant and native AgB preparations were highly reactive for human IgG4 antibodies in serum of cystic echinococcus (CE) patients. Recombinant AgB.MBP (rAgB.MBP) showed approximately 65% sensitivity in detection of IgG4 serum antibodies by ELISA from confirmed CE patients. Cross-reactivity (33%) occurred with alveolar echinococcosis (E. multilocularis) sera but recombinant AgB showed no seroreactivity with sera from other helminth infections tested (schistosomsis, onchocercsis, cysticercosis) or from uninfected individuals residing in CE endemic or non-endemic regions. The serologic sensitivity (63%) for IgG4 antibodies of a native AgB fraction enriched from human hydatid cyst fluid was similar to that for recombinant AgB (65%) though specificity was slightly lower (81%). A dot-ELISA for detection of total IgG, incorporating the rAgB.MBP resulted in 74% sensitivity and 88% specificity for human CE and 93% sensitivity and 65% specificity for native AgB. Recombinant AgB is a potential replacement for native antigens currently being used and could provide a better standardised E. granulosus specific test for clinical confirmation for CE especially for IgG4 antibody detection which appears to be predominantly associated with advanced disease.     

The use of monoclonal antibody for the immunodiagnosis of Fasciola gigantica infection in cattle

Antigens that were specific to Fasciola gigantica were obtained from the whole worm homogenate of the parasite by immunoaffinity chromatography in cyanogen bromide-activated sepharose 4B columns and used for the production of monoclonal antibodies. The F. gigantica-specific monoclonal antibody was labelled with horseradish peroxidase and used for the detection of circulating antigen by the direct ELISA method in the sera of cattle experimentally infected with the parasite. Circulating antigens were detectable in the sera of the animals as from the third week after infection while negative absorbance values were obtained 2 weeks after the termination of the infection by chemotherapy with oxyclozanide. This immunodiagnostic method offers an attractive alternative as a supplement to the conventional coprological diagnosis of fasciolosis.     

Role of magnetic resonance in hemodialysis patients with amyloid shoulder arthropathy]

Monoclonal antibodies (MAbs) were generated by immunizing mice with a truncated recombinant protein corresponding to the immunodominant region (residues 1-120) of hepatitis C virus (HCV) nucleocapsid protein. The specific recognition by either human sera or mouse monoclonal antibodies of overlapping peptides spanning the core region 1-120 as well as the comparison with epitopes described earlier allowed the fine mapping of HCV core. Within the region 1-120, the major antigenic domain could be restricted to the first 45 amino acids. Indeed, the peptide S42G (residues 2-45) allowed the detection of an anti-HCV core response by all anticore-positive human sera examined. According to their epitope localization, three groups of mouse MABs could be evidenced that were directed against different regions of core. Group II MAbs recognized a strictly linear epitope (QDVKF, residues 20-24), whereas group I MABs were directed against a conformational epitope mainly located at the amino acid residues (QIVGG, 29-33). The epitope of group III MABs was also conformational (PRGRRQPI, residues 58-65). These three epitopes appeared close but different from the three major human epitopes RKTKRNTN, VYLLPR, and GRTWAQPGYPWPLY (residues 7-17, 34-39, and 73-86, respectively). Group II MAB 7G12A8 and group I MAB 19D9D6 were used in a sandwich ELISA for the capture and the detection, respectively, of viral core antigen in sera of patients with chronic HCV infection. After treatment of sera with triton x 100 in acidic conditions, amounts of viral antigen as low as 20 pg/ml of sera could be detected.     

Selection of antigenic and immunogenic mimics of hepatitis C virus using sera from patients

Using sera from hepatitis C virus (HCV)-infected patients and noninfected subjects to screen random peptide libraries displayed on phage, we selected peptides specifically reacting with sera from infected patients. These phage- borne peptides were shown to mimic distinct HCV determinants. They detected in all cases the presence of anti-HCV Abs in a large panel of patients' sera, thus demonstrating the high sensitivity of the selected peptides as diagnostic markers. In addition, this diagnostic approach allowed a detailed characterization of the individual humoral response to viral infection. Phage-displayed HCV mimics were substitutes for the authentic HCV epitopes in inducing a strong specific response against HCV when used as immunogens in mice. These results support the search for HCV mimics with the potential to elicit a protective immune response as leads for the development of a mimotope-based vaccine against viral infection.     

Construction of a semisynthetic antibody library using trinucleotide oligos

A semisynthetic antibody library composed of single chain Fv fragments (scFv) was constructed by replacing the heavy chain CDR3 region of a human scFv by a random sequence of eight amino acids using trinucleotide codons. After cloning into a phage display vector, an antibody library was generated with a complexity of 8 x 10(8) independent clones. The library was screened for binders to dinitrophenol, fluorescein isothiocyanate and 3-nitro-4-hydroxy-5-iodophenylacetic acid. scFv antibodies that specifically bound the antigen were obtained in each case.     

Immune responses to the R4 protein antigen of group B streptococci and its relationship to other streptococcal R4 proteins

The R antigen, a trypsin-resistant protein observed in group A, C, F, G, and L streptococci, has also been found in group B streptococci (GBS). Although four species of the R antigen have been described for GBS, the R4 protein is the most prevalent in GBS isolates recovered from humans. This study examined the prevalence of antibodies against the R4 antigen by Western blot (immunoblot) (WB) in sera from 40 mothers colonized with GBS serotype II and III and from 26 noncolonized mothers; 92.5% of the colonized mothers had anti-R4 antibodies, compared with 54% of the noncolonized mothers (P < 0.001). Findings of antibodies in neonatal cord sera (n = 14) were concordant with maternal results by WB analysis for 71% of mother-infant pairs colonized with serotype II and for 57% of pairs colonized with serotype III. Of mothers known to be colonized with type II/R4 or III/R4, 100% (n = 12) had antibody against R4 by WB. This study also evaluated the prevalence of antibody to the GBS R4 antigen in 48 sera from individuals with high and low group A streptococcal anti-DNase B titers. Of those individuals with an anti-DNase B titer of > 640, 64% had a positive WB for anti-R4 antibody, compared with 30% of individuals with low anti-DNase B titers (P < 0.05). The R4 antigen of GBS had immunologic identity to the R4 antigen of group A streptococci. Overall, the findings suggested that antibodies to the streptococcal R4 antigen were commonly present in GBS-colonized mothers and that transplacental passage of these antibodies occurred. The presence of antibody to R4 in non-GBS-colonized individuals may be due to immunologic responses to past exposure to the R antigen present in GBS or other streptococcal groups.     

q analogs of the radial Schr?dinger equation in N space dimensions

Native parvovirus B19 was used as antigen to produce a mouse monoclonal antibody, R92F6, which reacted with B19 VP1 and VP2, neutralised the virus in bone marrow culture, and labelled infected cells in paraffin-embedded tissues from cases of B19-related fetal hydrops. The B19 epitope recognised by R92F6 (amino acids 328-344 from the amino terminal region of B19 VP2) appears to be highly conserved, since these tissue specimens were obtained over a 13 year period from widely spaced locations in the UK. This epitope was synthesised as a peptide (S7b) which was used as antigen to produce a mouse monoclonal antibody, 3H8, which specifically reacted with the B19 capsid proteins VP1 and VP2 in immunofluorescence and immunoblot assays. 3H8 was also capable of labelling formalin-fixed, paraffin-embedded, B19-infected fetal tissue and was shown to be of the same isotype as R92F6 (IgG1). Highly conserved epitopes derived from conserved amino acid sequences are valuable in the diagnosis of infectious disease. If these can be recognised and accurately synthesised, the production of specific mouse monoclonal antibodies may be possible for many human pathogens. Considering the vast amount of sequence data available in the literature, this approach seems to be both feasible and of wide potential.     

Reactivities of human sera with human herpesvirus-8-infected BCBL-1 cells and identification of HHV-8-specific proteins and glycoproteins and the encoding cDNAs

The reactivates of human sera with uninduced and phorbol ester (TPA)-induced human herpesvirus-8 (HHV-8)-infected BCBL-1 cells were examined by immunofluorescence assay (IFA) and by radioimmunoprecipitation reactions (RIP). The seroprevalence of HHV-8 infections is low in the United States general population and only low levels of HHV-8 antibodies were detected in the seropositive sera. In contrast, high levels of antibodies against HHV-8 lytic and latent antigens were detected by IFA in the sera from HIV+ Kaposi's sarcoma (KS)-positive individuals. These sera recognized several proteins and glycoproteins from BCBL-1 cells in RIP reactions. Two types of antibody responses were detected in the sera from HIV+ KS- homosexual men. In majority of the sera with and without detectable HHV-8 DNA in the peripheral blood mononuclear cells (PBMC), significantly low levels of HHV-8 antibodies were detected by IFA. These sera recognized only a subset of HHV-8 proteins and glycoproteins in RIP reactions. In contrast, in a subgroup of sera from HIV+ KS- homosexual men, higher levels of IFA antibodies against HHV-8 lytic and latent antigens were detected. These sera also recognized several viral proteins and glycoproteins in RIP reactions. These results suggest that antibody response profiles to HHV-8 infection vary significantly and serologic assays to detect antibody responses to a panel of both lytic and latent antibodies may be required for maximum sensitivity. Screening of a cDNA library from TPA-induced BCBL-1 cells with an HIV+ KS+ serum identified cDNAs encoding 12 HHV-8 proteins. Further characterization of these HHV-8 proteins would define the HHV-8 antigens useful for seroepidemiological studies and in discriminating lytic, latent, past, and/or reactivation infections.     

Replication of hepatitis C virus in cultured non-neoplastic human hepatocytes

We established a replication system for hepatitis C virus (HCV) using the PH5CH non-neoplastic human hepatocyte line that had been immortalized with simian virus 40 large T antigen. In cells inoculated with sera derived from two HCV-positive blood donors, positive-stranded HCV RNA was detected up to 30 days postinoculation (p.i.). Semi-quantitative analysis of HCV RNA revealed that HCV multiplied during the period of culture. Sequence analysis of the HCV hypervariable region 1 (HVR1) in both cases indicated that HVR1 populations from the cells at 8 days p.i. were apparently different from those of the original inocula. HVR1 populations in infected cells became homogeneous or just a few species were selected over time. These results suggest that HCV is replicating in the human hepatocyte PH5CH cells. This culture system will be useful for detailed studies of the biological effects of HCV in human hepatocytes.     

Gastric carcinoma in young adults

Among 47 blood donors tested positive with HCV EIA 2.0 Abbott, 27 (57.4%) also reacted with four ?third-generation' EIAs. The presence of anti-HCV antibodies was confirmed with 3 different immunoblot assays in 16 of 27 sera (34.0%) while 10 samples (21.3%) had indeterminate profile with antibodies usually directed against structural core antigen. Anti-HCV core IgM response was found in 12 of 47 sera (25.5%) and HCV viremia detected by the polymerase chain reaction (PCR) procedure was observed in 15 samples (31.9%). A comparative study of the different markers confirmed a good correlation between a strong antibody response in EIAs and immunoblot assays and the presence of HCV RNA in the serum; only 2 immunoblot indeterminate samples were PCR positive. An association was observed between IgM antibodies against "core' epitopes and HCV RNA carriage: all IgM-positive sera were found positive by PCR. However, the direct detection of viral genome remains the best method for identifying HCV carriers in the blood donor population.     

Functional and molecular characterization of human monoclonal antibody reactive with the immunodominant region of HIV type 1 glycoprotein 41

The immunoreactivity, functional activity, and molecular features of a human monoclonal antibody (HMAb), F240, from an HIV-1-infected individual have been studied. Flow cytometric analysis demonstrated that F240 is reactive with cells infected with a broad range of laboratory isolates but not with uninfected cells. Reactivity of F240 is greatly enhanced by preincubation of infected cells with soluble CD4, and to a much lesser extent, with F105, an HMAb reactive with the CD4-binding site of gp120. This enhancement is temperature dependent, with maximum enhancement observed at 37 degrees C, and suggests that the F240 epitope may be more accessible after gp120 has bound to CD4 in vivo. Immunoblot analysis reveals antigen specificity of F240 for gp41 or its precursor gp160. F240 specificity is mapped to the immunodominant region of the gp41 ectodomain by Pepscan analysis. This epitope has been implicated in eliciting nonprotective antibodies that enhance infection in the presence of complement. Consistent with this, F240 failed to neutralize laboratory isolates and enhanced viral infection in a complement-dependent manner. The F240 VH demonstrates extensive somatic mutations compared with the product of its closest homologous germline gene VH3-3.11. Most amino acid substitutions occur in CDR2, characteristic of an antigen-driven response, and in FR3, a phenomenon observed in other anti-HIV-1 envelope HMAbs. Primary structure analysis of the F240 heavy chain revealed strong homology in the CDR domains to an HMAb (3D6) reactive with the same gp41 region, which suggests that these HMAbs could define a potential human antibody clonotype.     

Anti-idiotypic activity against anti-myeloperoxidase antibodies in pooled human immunoglobulin

We investigated the ability of six different pooled human immunoglobulin (PHIG) preparations to inhibit the binding of anti-myeloperoxidase (MPO) antibodies to MPO. All six PHIG preparations inhibited the binding of anti-MPO antibodies from six sera to MPO in a concentration-dependent manner in the concentration range 0.016-10 mg/ml. There was considerable variation in the ability of each PHIG preparation to inhibit the binding of anti-MPO antibody in a given serum. Further differences were seen in the ability of a given PHIG to inhibit anti-MPO binding in different sera. F(ab')2 fragments from two PHIG preparations also inhibited in a concentration-dependent manner anti-MPO binding to MPO in all six sera in the concentration range 0.002-2.65 mg/ml, with a maximum inhibition of 42%. Little inhibition was seen with F(ab')2 of normal human IgG from individual donors (1.8-12.2% at the maximum concentration of 2 mg/ml). F(ab')2 fragments from three anti-MPO containing sera and two affinity-purified anti-MPO antibodies were eluted by affinity chromatography from Sepharose-bound PHIG F(ab')2 and showed anti-MPO antibody activity. We have shown that PHIG and F(ab')2 fragments of PHIG inhibit anti-MPO binding to MPO, and further that F(ab')2 fragments of PHIG bind to F(ab')2 fragments of anti-MPO antibodies. These observations indicate binding between the variable regions of PHIG and the antigen binding site of anti-MPO antibodies, and are consistent with an anti-idiotypic reaction. The variability seen in the inhibitory effect of the different PHIG preparations in anti-MPO-positive sera implies differences in their anti-idiotype content, while the variability of the inhibitory effect of a particular PHIG preparation between different sera suggests heterogeneity in the idiotypic repertoire of anti-MPO antibodies. Such variations in the inhibitory effect of different PHIG preparations on antibody binding may be an important determinant of their therapeutic effect.     

Isotype-specific immune response to a single hepatitis C virus core epitope defined by a human monoclonal antibody: diagnostic value and correlation to PCR

In this study we tested the seroreactivity of 223 selected anti-HCV-reactive blood donors to the human B-cell epitope N-VYLLPR-C (C34-39) of the hepatitis C virus core antigen. The epitope was recently identified and characterized by the human monoclonal IgG antibody Ul/F10 and is located within the amino acid residues 34-39 of the aminoterminal core region. The blood donor sera were selected from anti-HCV ELISA (Ortho, 2nd generation)-reactive samples. Sixty-seven of these sera were further reactive in RIBA (Ortho, 2nd generation). According to their RIBA pattern, these samples were divided into four groups. Samples in the first group (n = 18) reacted to all four recombinant HCV antigens. The samples of the second (n = 9) and third group (n = 8) reacted to c22-3/c33c and c22-3/c100-3, respectively. Sera from group 4 (n = 32) showed a RIBA indeterminate pattern with reactivity only to c22-3. All 223 samples were analyzed for anti-C34-39 antibodies by ELISA, and the 67 RIBA-reactive samples were additionally tested for the presence of HCV RNA by RT/PCR. In groups 1 and 2, over 80% of the samples showed anti-C34-39 reactivity which was restricted to the IgG1 isotype. In contrast, in groups 3 and 4, antibodies to the epitope C34-39 were detected in less than 10% of the samples. Interestingly, the anti-C34-39 response correlates with the presence of HCV RNA; 95.5% of the samples had coincident results in all subgroups. None of the RIBA-negative sera showed a specific seroreaction to the C34-39 peptide.     

Diverse Fab specific for acetylcholine receptor epitopes from a myasthenia gravis thymus combinatorial library

The muscle weakness in myasthenia gravis (MG) is caused by heterogeneous high-affinity IgG autoantibodies to the nicotinic acetylcholine receptor (AChR), a complex ion channel glycoprotein. These antibodies are clearly responsible for reducing AChR numbers at the neuromuscular junction in myasthenia; however, the origins, diversity, specificity and pathogenicity of individual antibodies have not yet been established. We have cloned and characterized four different AChR-specific Fab from an MG patient's thymus by screening an IgG1/kappa gene combinatorial lambda phage library with soluble human AChR labeled with [125I] alpha-bungarotoxin. Unlike most previously cloned human antibodies, all four Fab immunoprecipitated soluble human muscle AChR. Two Fab strongly inhibited binding of mAb to the main immunogenic region on the alpha subunits and one Fab bound to an epitope on the fetal-specific gamma subunit. In sensitivity and fine specificity, these Fab resembled the anti-AChR antibodies found in many MG patients, including the donor. The closest germline counterparts for their heavy chains were in VH families 1, 3 and 4; however, there were many differences consistent with an antigen-driven response of diverse B cell clones. The combinatorial approach holds promise for further analysis of human autoantibodies.