Probing the idiotype/anti-idiotype antibody interaction with a set of synthetic peptide homologues.

Anti-idiotypic (anti-Id) antibodies were raised against two murine monoclonal antibodies (mAb 1/1 and mAb 2/1) which recognise two distinct and well-characterised epitopes on a 24-residue synthetic peptide representing part of the haemagglutinin (HA) of influenza virus. A monoclonal anti-Id antibody, specific for mAb 2/1, could bind to mAb 2/1 when the paratope of the latter was occupied with peptide, indicating that this anti-Id antibody is directed to a framework idiotope. In contrast, an anti-Id mAb derived from mAb 1/1-immunised mice was inhibited in its binding to Id by the parent peptide and also by the heptapeptide NVPEKQT which constitutes the epitope recognised by mAb 1/1. The small size of this synthetic peptide eliminates the possibility of significant steric inhibition in the system, and establishes that this mAb is a true paratope-directed anti-Id antibody. The interaction of this anti-Id mAb with the paratope of mAb 1/1 in the presence of a set of peptide homologues of the epitope was also examined. A peptide as short as 5 residues, which contains two of the three irreplaceable residues of the epitope, could inhibit binding between the two mAbs.     

Anti-idiotypic antibodies of a predefined specificity generated against CDR3VH synthetic peptides define a private anti-CD4 idiotype

A synthetic peptide corresponding to the third complementarity determining region (CDR) of the heavy chain (CDR3VH) of anti-Leu3a, a monoclonal anti-CD4 antibody which inhibits HIV gp120 binding to CD4, was used to elicit specific anti-peptide antibodies in rabbits. The anti-peptide antisera showed anti-idiotypic antibody (anti-Id) activity and recognized both the immunizing peptide and the intact cognate protein by ELISA. In addition, the antisera reacted with isolated heavy chains of anti-Leu3a by Western blot analysis. The lack of reactivity with a panel of monoclonal anti-CD4 antibodies suggested that the anti-peptide antisera recognize a private idiotype (Id) associated with the anti-Leu3a CDR3VH region. Further studies demonstrated the inability of the rabbit antisera to inhibit the binding of anti-Leu3a to the CD4 molecule. In addition, soluble recombinant CD4 was unable to inhibit the binding of the rabbit anti-peptide antisera to anti-Leu3a indicating that the CDR3VH region may not be involved in CD4 recognition. Anti-Id containing sera from mice, rabbits and nonhuman primates immunized with the intact anti-Leu3a molecule did not bind the CDR3VH synthetic peptide, suggesting that the corresponding region of anti-Leu3a may not represent an immunodominant idiotypic determinant in thes e species. These results suggest the potential use of synthetic peptides corresponding to immunoglobulin variable (V) region amino acid sequences in generating anti-Id reagents of a predefined specificity. In addition, V-region synthetic peptides may be useful in mapping the idiotopes recognized by an anti-Id response to the cognate molecule.     

Characterization of private and cross-reactive idiotypes associated with human antibodies to hepatitis B surface antigen.

Four mouse monoclonal anti-idiotypic antibodies (anti-Id) were generated against human monoclonal and polyclonal antibodies to hepatitis B surface antigen (HBsAg). These monoclonal anti-Id, along with a polyclonal anti-Id raised in rabbits, were used to characterize the idiotype (Id) specificity of the human antibody response to HBsAg (anti-HBs). The anti-Id reagents identified distinct private and cross-reactive Id expressed on monoclonal and polyclonal human anti-HBs preparations respectively. The anti-Id recognized both HBsAg combining site and non-combining site related private Id, and HBsAg combining site related cross-reactive Id. The Id specificities recognized by two of the monoclonal anti-Id were associated with the H chain alone, whereas two of the monoclonal anti-Id, along with the polyclonal anti-Id appeared to recognize Id determinants associated with both isolated H and L chains. These data suggest that Id heterogeneity exists within the human antibody response to HBsAg. The knowledge that Id heterogeneity exists is of importance in understanding the observed variability in the immune response during hepatitis B virus infection.     

A Murine Monoclonal Anti-Idiotypic Antibody Detects a Common Idiotope on Human, Mouse and Rabbit Antibodies to Allergen Lol p IV

A syngeneic mouse monoclonal anti-idiotypic antibody (anti-Id), designated as B1/1, was generated against a monoclonal antibody (MoAb 91) specific for Ryegrass pollen allergen Lol p IV. This anti-Id recognized an idiotope (Id) that was also present on other monoclonal antibodies with the same specificity as MoAb 91. Observations that (i) the anti-Id inhibited the binding of MoAb 91 to Lol p IV and (ii) the Id-anti-Id interaction could be inhibited by Lol p IV indicated that the Id was located within or near the antigen combining site. These properties served to characterize B1/1 as an internal image anti-Id. Evidence that an immune response in different species to Lol p IV elicits the formation of antibodies which express a common Id was provided by the observations that (i) the Id-anti-Id interactions could be inhibited by mouse, human and rabbit antisera to Lol p IV and (ii) the binding of these antisera to Lol p IV could be inhibited by the anti-Id. Interestingly, the internal image anti-Id B1/1 also recognized an Id on a monoclonal antibody which was directed to an epitope of Lol p IV, different from that recognized by MoAb 91.     

Detection of antibody, idiotype, and anti-idiotype forming cells by in situ immunocytochemical staining

Methods are described for the immunocytochemical staining of cryostat sections of lymphoid tissue with enzyme conjugates of antigen, idiotype (Id) and anti-idiotype. Results established this as a useful approach, for simultaneously detecting Id and anti-Id antibody forming cells (AFC) in situ. As a model, the 5AF6 Id family associated with the BALB/c mouse antibody response against the p-azophenyl-arsonate (Ar) epitope was examined by two-color immunocytochemical staining, allowing the simultaneous detection of both Id+ and Id- anti-Ar AFC. Spleens from mice secondarily immunized with Ar antigen but not normal mice contained anti-Id AFC stained with the 5AF6 Id but not with another immunoglobulin of the same isotype. A sequential staining method was developed which allowed the detection of both Id and anti-Id AFC in the same tissue, thus providing a means of examining Id and anti-Id antibody networks in intact lymphoid tissues.     

Anti-idiotypic antibody-induced protection against Clostridium perfringens type D.

A monoclonal antibody (BALB/c mouse) with specificity for a neutralizing epitope on the epsilon-toxin produced by Clostridium perfringens type D was used to raise anti-idiotypic antibodies (anti-Id) in different strains of mice and rabbits. These were purified and used in cross-immunization studies to induce anti-(anti-idiotype). All strains of mice and rabbits immunized with BALB/c-derived anti-Id showed a high-titer antibody response directed towards the active site of the toxin. This protected the animals against toxin challenge and against an oral dose of the vegetative organisms. Animals immunized with other anti-Id preparations showed no specific antibody response and were not protected. Guinea pig peritoneal macrophages have a cell surface receptor for the toxin, and incubation of these cells with BALB/c anti-Id allowed them to survive toxin challenge, indicating that occupation of the receptors by the anti-Id prevented binding by the toxin. In conclusion, we have shown that an internal-image anti-Id preparation will induce protective immunity in syngeneic and xenogeneic animals and furthermore that immunity to a single epitope on the exotoxin is sufficient to protect against the toxin and clinical sequelae evoked by the disease-causing organism itself.     

抗聚合人血清白蛋白的抗独特型抗体

一种针对兔抗聚合人血清白蛋白抗体(anti-PHSA,Ab_1)的抗独特型抗体(anti-anti-PHSA,Ab_2),已通过免疫同种动物制成。该Ab_2具有以下特性:(1)它既可与兔anti-PHSA反应,也可与鼠的单克隆的anti-PHSA反应。(2)它与鼠的单克隆的anti-PHSA的反应,可受到PHSA和乙型肝炎病毒(HBV)上的聚合人血清白蛋白受体(PHSA-R)的抑制。(3)它可在异种动物体内诱导产生具有anti-PHSA活性的抗-抗独特型抗体(anti-anti-anti-PHSA,Ab_3),而且在收获的Ab_2血清中也查见了Ab_3。本研究不仅表明了针对PHSA的免疫应答中独特型网络调节的客观存在,而且在其抗独特型抗体(anti-Id)中,的确存在具有PHSA内映像的成分。     

Characterization of a novel human immunodeficiency virus type 1 neutralizable epitope within the immunodominant region of gp41

Previously, we generated human monoclonal antibodies using peripheral blood mononuclear cells from an asymptomatic human immunodeficiency virus type 1 (HIV-1)-seropositive donor. One of these monoclonal antibodies (designated clone 3, CL3) recognized 10 amino acids (GCSGKLICTT) within the immunodominant region (cluster I) of the transmembrane envelope glycoprotein gp41 and neutralized infection of target cells with different laboratory isolates. Because the epitope recognized by CL3 has two cysteine residues that could potentially produce a disulfide loop in gp41, we analyzed binding of our monoclonal antibody to the cyclic and linear motif of the peptide sequence IWGCSGKLICTTAVP (residues 600-614). The CL3 antibody did not bind to the synthetic cyclic peptide but did recognize the linear form. Two polyclonal rabbit sera against both the linear and cyclic peptides were then generated. Both antisera bound to viral glycoproteins gp41 and gp160, but neither sera neutralized HIV-1 laboratory isolates. Using a set of alanine-substituted IWGCSGKLICTTAV peptides, we analyzed binding of polyclonal antisera and CL3. The profile of binding of polyclonal antisera to these peptides was different from that of CL3 to the same peptides. This suggests that CL3 recognized a unique neutralizable core epitope, which was not immunogenic in either the cyclic or the linear IWGCSGKLICTTAVP peptides used as immunogens in the rabbits.     

Idiotypic cascades associated with the CD4-HIV gp120 interaction: principles for idiotype-based vaccines.

Idiotypes (Id) are antigenic determinants expressed on the variable (V) region of the immunoglobulin molecule. Id-bearing antibodies, or Ab-1, are produced upon stimulation with a given antigen. Ab-1 may elicit the production of anti-idiotypic antibodies (anti-Id) or Ab-2. The anti-Id also expresses Id determinants and may in turn elicit the production of anti-anti-Id or Ab-3. The production of Ab-1, Ab-2, and Ab-3 responses resulting from stimulation with the antigen is representative of components within an Id cascade. The existence of this Id cascade is the basis for the development of Id based strategies for controlling the immune response to infectious agents and tumors. In this review we will focus on several aspects regarding the Id cascades that may be operational during the immune response to the human immunodeficiency virus (HIV). In light of several studies which suggest the existence of Id-anti-Id interactions operating during the course of HIV infection, we will discuss the potential applications of Id based strategies in manipulating the immune response to HIV.     

Idiotypic Cascades Associated with the CD4-HIV gpl20 Interaction: Principles for Idiotype-Based Vaccines

Idiotypes (Id) are antigenic determinants expressed on the variable (V) region of the immunoglobulin molecule. Id-bearing antibodies, or Ab-1, are produced upon stimulation with a given antigen. Ab-1 may elicit the production of anti-idiotypic antibodies (anti-Id) or Ab-2. The anti-Id also expresses Id determinants and may in turn elicit the production of anii-anti-Id or Ab-3. The production of Ab-1, Ab-2, and Ab-3 responses resulting from stimulation with the antigen is representative of components within an Id cascade. The existence of this Id cascade is the basis for the development of Id based strategies for controlling the immune response to infectious agents and tumors. In this review we will focus on several aspects regarding the Id cascades that may be operational during the immune response to the human immunodeficiency virus (HIV). In light of several studies which suggest the existence of Id-anti-Id interactions operating during the course of HIV infection, we will discuss the potential applications of Id based strategies in manipulating the immune response to HIV.     

Study of idiotypes expressed by monoclonal antibodies to the 35 kD and 12 kD antigens of Mycobacterium leprae.

Rabbit antisera were raised against four monoclonal antibodies (MoAb) binding with the 35 kD protein and four MoAb binding with the 12 kD protein antigen of Mycobacterium leprae. Antisera showed idiotype (Id) specificity following cross-absorption with normal mouse globulin. One Id on a single MoAb and another Id shared between three MoAb were identified for each group. Functional studies were carried out with the Rb04 anti [anti-35 kD] specificity. The expression of this Id and paratope in antigen immunized mice was associated with Igh alleles. Inoculation of mice with anti-Id Rb04 induced an 'Ab3' serum response of corresponding Id specificity only when the anti-Id was given in emulsion with incomplete Freund's adjuvant (IFA). Conversely, prior injection of soluble anti-Id inhibited the subsequent Ab3 response to Rb04/IFA. Moreover, the suppressive effect of soluble anti-Id was abrogated by prior injection of 50 mg/kg cyclophosphamide. These results indicate that regulatory mechanisms similar to those involved in antigenic stimulation may explain the stimulatory or suppressive potency of anti-Id antibodies. Finally, the Ab3 responses to the two tested anti-Ids did not contain any antigen binding activity.     

A central anti-DNA idiotype in human and murine systemic lupus erythematosus

The monoclonal anti-DNA autoantibody A52 (IgG2b) was obtained from a (NZB X NZW)F1 (B/W) hybridoma. Two rabbits were immunized with the pure monoclonal antibody and produced anti-idiotypic (Id) antibodies. The purified anti-Id reacted with three different B/W monoclonal anti-DNA antibodies at or close to their DNA binding sites. Moreover, the rabbit antibodies had a profound inhibitory effect on the polyclonal anti-DNA activity in the majority of sera derived from B/W mice and human systemic lupus erythematosus (SLE) patients. The A52 IgG must, therefore, represent a major cross-reactive Id of anti-DNA immunoglobulins. In addition, the rabbit anti-Id antibodies may act as the "internal image" of antigen and should prove useful in modulation of the autoimmune response to DNA in SLE.     

Expression of two cross-reactive idiotypes on mouse antibodies against bromelain-treated mouse erythrocytes.

Two cross-reactive anti-idiotype (Id) antibodies were previously prepared from sera of rabbits immunized with mouse monoclonal antibodies against bromelain-treated mouse erythrocytes (BrMRBC). Most of the anti-BrMRBC plaque-forming cells (PFC) were suppressed by either of the two anti-Id antibodies. The Id profiles of anti-BrMRBC PFC were almost identical among various cell populations in a strain, but different among various mouse strains. Mouse sera contained both of the Id-bearing immunoglobulins Ig, and a significant part of the Id-bearing Ig were eliminated by absorption with BrMRBC. Nude BALB/c mice were almost equal to normal BALB/c mice in the Id patterns of anti-BrMRBC PFC and in the concentrations of the Id-bearing Ig. The injections of anti-Id antibodies into suckling mice suppressed, specifically, the development of the B cells to produce the homologous Id-bearing Ig, but the injection of Id-bearing monoclonal antibodies barely affected Id expression. It is suggested that the two Id are encoded in germ-line genes of mice, and are expressed independently of each other and Id-anti-Id regulations by T cells or B cells.     

Epitope-vaccines: a new strategy to induce high levels of neutralizing antibodies against HIV-1

Based on the experimental evidence that gp120 subunit vaccine did not protect individuals from HIV-1 infection, we suggested that epitope-vaccines of HIV-1 gp41 may be a new strategy to induce high levels of neutralizing antibodies against HIV-1, and characterised immunogenicity of epitope-vaccines. Two epitopes, RILAVERYLKD-epitope (aa586-596) on the N-domain and ELDKWA-epitope (aa669-674) on the C-domain of gp41, were demonstrated by us and others to induce protective activity. After vaccination course, the RILAVERYLKD-dimer epitope-vaccine [C(RILAVERYLKDG)2-BSA] induced strong epitope-specific antibody response by about 1:25,600 dilution, and the ELDKWA-tetramer epitope-vaccine [C-(ELDKWAG)4-BSA] could yet induce strong antibody response to ELDKWA-epitope by 1:12,800-25,600 dilution of antisera in mice, while rgp41 subunit vaccine induced very weak antibody response to both epitopes (1:400). In rabbit experiments, the titres of ELDKWA-epitope-specific antibody induced by ELDKWA-epitope-vaccine [C-(ELDKWAG)4-BSA] reached to 1:6,400, while rgp41 subunit vaccine induced very weak antibody response to this epitope and to P1 and P2 peptides (1:400). Moreover, the ELDKWA-epitope-specific antibodies in mice and rabbit antisera induced by epitope-vaccine could very strongly interact with P2 peptide sequence-corresponding to the C-domain of gp41 (dilution by 1:25,600), and the RILAVERYLKD-epitope-specific antibodies in mice antisera induced by epitope-vaccine could also very strongly interact with P1 peptide sequence-corresponding to the N-domain of gp41 (dilution by 1:102,400). All these results provided experimental evidence that epitope-vaccine may be a new general strategy to induce high levels of neutralizing antibodies against HIV-1 or other viruses.     

Depletion in antibodies targeted to the HR2 region of HIV-1 glycoprotein gp41 in sera of HIV-1-seropositive patients treated with T20

The anti-HIV drug T20 is a synthetic peptide derived from the HR2 region of HIV-1 gp41. T20 contains the sequence ELDKWA, which binds the broadly neutralizing antibody 2F5. Using plates coated with T20 or with synthetic peptides and recombinant proteins representing gp120 or gp41 domains, this study investigated by enzyme-linked immunosorbent assay the levels of antibodies directed to the gp160 molecule in patients treated with T20. Analysis of sera obtained before and after administration of T20 indicated that the levels of antibodies directed to T20, to MBP44, a maltose binding protein representing the HR2 region, and to 4765, a synthetic peptide containing the sequence ELDKWA, fell following administration of T20, while the levels of antibodies directed to other regions of gp41 ectodomain and to gp120 remained stable. The decline observed was independent of the viral load and of the total IgG concentration. Follow-up studies with sera obtained from HIV-1-seropositive patients naive to T20 indicated no decline in the level of antibodies directed to HR2 and other regions of gp160. Analysis of sera obtained from a patient after 2 months of T20 treatment interruption showed a level of antibodies to the HR2 region similar to that measured before administration of T20. The addition of increasing amounts of T20 to sera from T20-naive patients decreased the level of serum antibodies against peptide 4765, T20, and MBP44. The observation of antibody depletion by T20 suggests that anti-gp41 antibodies may interfere with T20 treatment by forming T20-antibody complexes.     

Human monoclonal antibody 98-6 reacts with the fusogenic form of gp41

A mixture of two peptides from gp41 (N36 and C34) forms an alpha-helical structure that is thought to represent the fusogenic form of gp41. A human anti-gp41 monoclonal antibody (mAb 98-6), generated from the cells of an infected individual, reacted poorly with C34, but binding was strongly enhanced when N36 was added, indicating that the mAb reacts with a conformational epitope present in the fusogenic structure formed by the interaction of peptides N36 and C34. The epitope recognized by mAb 98-6 was found in lysates of virions on oligomeric forms of gp41 (dimers, trimers, and tetramers). On infected cells, the epitope was present as oligomers of gp41, as monomers of gp41, and as part of the envelope polyprotein gp160, obtained after biotinylation of intact cells, which were then lysed and immunoprecipitated with various mAbs. In lysates of infected cells, the epitope was present as part of both monomeric gp41 and gp160. These studies demonstrate that infected humans can respond to the fusogenic form of gp41 and that the anti-gp41 mAb studied here recognizes a conformational epitope formed by the interaction of two regions of gp41, which forms an alpha-helical bundle. This epitope is found on several forms of gp41 as it occurs in virions, on the surface of infected cells, and in infected cells.     

Isolation and characterization of monoclonal antibodies elicited by trimeric HIV-1 Env gp140 protein immunogens

Eleven anti-HIV Env monoclonal antibodies (MAbs) were isolated from mice immunized with soluble Env proteins derived from the clade B Env, SF162, or DeltaV2 (a derivative of SF162 lacking the V2 loop). All six anti-gp120 MAbs studied, neutralized SF162 and their activities were dependent by the glycosylation patterns of the V1, V2 or V3 loops. Only one anti-gp120 MAb (an anti-V3 MAb) displayed cross-neutralizing activity, which was influenced by the type of V1 loop present on the target heterologous viruses. None of the five anti-gp41 MAbs studied displayed anti-SF162 neutralizing activity. Our studies indicate that the current limitation of soluble HIV Env gp140 immunogens to elicit robust cross-reactive neutralizing antibody responses is not only due to the elicitation of high titers of homologous antibodies but also due to the elicitation of antibodies whose epitopes are naturally occluded, or not present, on the virion-associated Env.     

Structural characterization of viral neutralizing monoclonal antibodies to hepatitis B surface antigen

In this study we describe the viral neutralizing activity of murine monoclonal antibodies (MAb) specific for hepatitis B surface antigen (HBsAg). This viral neutralizing activity was assessed in vitro by employing Hepatitis Delta Virus (HDV) and human hepatocytes as target cells. To further characterize these viral neutralizing antibodies we generated a panel of anti-idiotypic (anti-Id) reagents and serologically characterized these antibodies for epitope specificity, Id specificity, and Id heterogeneity. Direct binding and competitive inhibition solid phase enzyme immunoassay have demonstrated that two murine MAb specific for HBsAg (anti-HBs), designated A1.2 and A3.1, recognize similar or overlapping epitopes on HBsAg, while monoclonal anti-HBs, designated A2.1 recognizes a unique HBsAg epitope. Further, Id analysis using monoclonal and polyclonal anti-Id reagents have identified both a private and a cross-reactive Id, respectively, on the anti-HBs, A1.2 preparation. The source of the idiotypic cross-reactivity between A1.2 and A3.1 has been identified, using Western blot analysis, to conformational determinants expressed by the heavy (H) and light (L) chains of these monoclonal anti-HBs. Lastly, the intrastrain antibody repertoire induced following HBsAg immunization was found to be relatively restricted in heterogeneity by clonotype analysis using isoelectric focusing and affinity immunoblot analysis. Interspecies variability in the anti-HBs response was observed based on epitope recognition using purified anti-HBs from a variety of species.     

HIV-1 gp41 by a common immunological epitope induces increased levels of antibodies against human interferon-β in HIV-1 positive individuals

Based on our finding that a similar epitope exists between human IFN-β (aa128–134) and HIV-1 gp41 (aa586–595), we examined 20 sera from healthy and 20 from HIV-1 infected individuals for IFN-β antibody levels by ELISA. The levels of anti-IFN-β antibody in sera from HIV-infected individuals were increased by about 160% in comparison with HIV-negative. We affinity-purified anti-gp41 antibodies from sera of HIV-1-infected individuals using rsgp41-sepharose column. One of three antibodies could recognize human IFN-β in comparison with antibodies from serum of a healthy individual. A mouse antiserum to human IFN-β recognized rsgp41 (recombinant soluble gp41, Env amino acid 539–684), while the normal mouse serum (pre-immune serum) did not bind to rspg41. These results indicate that a common immunological epitope exists between human IFN-β and HIV-1 gp41. The sequence-similarity suggests that this common immunological epitope may be located in the region aa128–134 of human IFN-β and the immunosuppressive domain (aa583–599) of HIV-1 gp41. The increased levels of antibodies against interferon-β in HIV-1 positive individuals may be explained by a common immunological epitope on human IFN-β and HIV-1 gp41.     

Towards an idiotype vaccine against mammary tumors. Induction of an immune response to breast cancer-associated antigens by anti-idiotypic antibodies

Previously we have reported on the production of two sets of human monoclonal antibodies reacting with mouse mammary tumor virus (MMTV) and human mammary tumor virus (HuMTV) cross-reacting antigens. B11 is a monoclonal IgG generated by the human-human hybridoma technique using axillary lymph node of breast cancer patients. 4.6/6 is a monoclonal IgG established by the mouse-human hybridoma procedure using peripheral blood lymphocytes of a healthy investigator working with the breast cancer cell line T47D which secretes HuMTV antigens. Two anti-idiotypic (Id) antibodies were produced by immunizing rabbits with B11 or 4.6/6. Following exhaustive adsorption on unrelated human-Ig, fetal calf serum and purification on B11 or 4.6/6 affinity columns, the anti-Id antibodies were shown to react specifically with their respective Id. The binding of these anti-Id antibodies to the respective Id was specifically inhibited by prior incubation of the Id with MMTV and/or HuMTV antigens. Rabbit anti-B11 and rabbit anti-4.6/6 anti-Id antibodies were employed to immunize female C3Heb mice. Following immunizations, humoral and cellular immune responses to MMTV-related antigens could be demonstrated. The sera of the mice contained anti-MMTV antibodies and delayed-type hypersensitivity was specifically expressed when irradiated cells of the Mm5MT line (which carry surface MMTV antigens) were injected. Our results support the notion that anti-Id antibodies harboring the internal image can immunize animals against tumor cells bearing on their surface viral-associated antigens.