Bovine monoclonal anti-idiotypes induce antibodies specific for a synthetic peptide bearing a neutralizing epitope of bovine herpesvirus 1 glycoprotein gI (gB).

Bovine monoclonal anti-Id mimicking a neutralizing epitope of bovine herpesvirus-1 (BHV-1) glycoprotein gI were developed. An epitope present on the 74K subunit of gI identified by a murine mAb 1E11 was selected for this study. Bovine lymphocytes from the prefemoral lymph node of a heifer immunized with mAb 1E11 were fused with SP-2/0, a nonsecreting murine cell-line. Two bovine x murine hybridomas secreting bovine monoclonal anti-Id specific for the Id of 1E11 were stabilized. These anti-Id inhibited the binding of 1E11 to purified glycoprotein gI in a dose-dependent fashion. Naive mice immunized with the anti-Id produced anti-anti-Id (Ab3) that reacted with BHV-1 glycoprotein gI in a RIA, and neutralized BHV-1 infection in vitro. The Ab3 also showed reactivity to the 74K subunit of authentic gI glycoprotein in a Western blot analysis, and to the synthetic peptide bearing the 1E11 epitope in a RIA. These results substantiate the presence of the population of anti-Ab2 that functionally resemble antibodies specific for the immunizing Ag BHV-1 in Ab3, and demonstrate the ability of these anti-Id to elicit BHV-1-specific antibody response.     

Induction of delayed-type hypersensitivity responses by monoclonal anti-idiotypic antibodies to tumor cells expressing carcinoembryonic antigen and tumor-associated glycoprotein-72

The use of anti-idiotypic antibodies as immunogens represents one potential approach to active specific immunotherapy of cancer. Two panels of syngeneic monoclonal anti-idiotypic antibodies were generated. One panel was directed against mAb CC49 and the other to mAb COL-1. mAb CC49 recognizes the pancarcinoma antigen (Ag), tumor-associated glycoprotein-72 (TAG-72), and mAb COL-1 recognizes carcinoembryonic antigen (CEA). Seven anti-idiotypic (AI) antibodies (Ab2) designated AI49-1–7 were generated that recognize the variable region of mAb CC49. These mAb were shown to inhibit the interaction of mAb CC49 (Ab1) with TAG-72 (Ag). Five anti-idiotypic antibodies designated CAI-1–5 were also generated to the anti-CEA mAb, COL-1 (Ab1). These Ab2 were shown to inhibit the interaction between COL-1 (Ab1) and CEA (Ag). Immunization of mice, rats, and rabbits with Ab2 directed against CC49 or COL-1 could not elicit specific Ab3 humoral immune responses, i.e., antibody selectively reactive with their respective target antigens. However, immunization of mice with the CC49 anti-idiotypic antibody (Ab2), designated AI49-3, could induce a delayed-type hypersensitivity response (DTH) specific for tumor cells that express TAG-72. Similarly, immunization of mice with an anti-idiotypic antibody directed against COL-1, designated CAI-1, could induce specific DTH cell-mediated immune responses to murine tumor cells that express human CEA on their surface. These results thus demonstrate that while some anti-idiotype mAb may not be potent immunogens in eliciting Ab3 humoral responses, they are capable of eliciting specific cellular immune responses against human carcinoma-associated antigens. This type of mAb may ultimately be useful in active immunotherapy protocols for human carcinoma.Some of the studies described in this paper were in partial fulfillment of requirements for the completion of Dr. Irvine's dissertation at the George Washington University     

Angiotensin II (AII)-related idiotypic network. I. Common occurrence of AII internal image on rabbit anti-idiotypic antibodies

Internal images of foreign Ag have been demonstrated in a variety of systems as anticipated by the idiotypic network theory formulated by Jerne. However, they seem to be of rare occurrence. In order to estimate the actual frequency of antibodies bearing internal images (Ab2-beta) of angiotensin II (AII), a phylogenetically conserved peptide made up of eight amino acids, nine rabbits were immunized with affinity or protein A purified anti-AII antibodies (Ab1) from allotype-matched rabbits. Four of nine antiidiotypic antibodies (Ab2) exhibited internal image-like reactivity. They recognized all the polyclonal Ab1 tested, whatever the species (rabbit, mouse, guinea pig). In addition, they were strongly reactive with three mAb specific for a carboxy terminus epitope on AII (mAb 110, 199, and 211) and with a fourth monoclonal Ab1 (133) identifying a more central epitope. Advantage was taken of this reactivity with mAb1 to purify Ab2-beta by affinity chromatography of Ab2 on Sepharose 4B covalently linked to the three monoclonal Ab1 specific for the carboxy terminus epitope. The eluate displayed typical internal image properties: 1) it reacted with all the polyclonal Ab1 tested, 2) this reaction was completely abolished by AII, and 3) rabbits and mice immunized with the eluate all produced Ab1. The AII related idiotypic network is thus characterized by high frequency and immunogenicity of AII internal images. In addition, reactivity of the latter with monoclonal Ab1 indicates variable expression on Ab2-beta of the epitopes defined by the mAb on the nominal Ag.     

Monoclonal anti-idiotypic antibodies induce humoral immune responses specific for simian virus 40 large tumor antigen in mice.

Mouse monoclonal anti-Id antibodies were generated against a mouse mAb (Ab-1) preparation specific for SV40 large tumor Ag (T-Ag). Four monoclonal anti-Id preparations each inhibited the binding of the monoclonal anti-SV40 T-Ag Ab-1 preparation to SV40 T-Ag. These anti-Id preparations appeared to recognize similar idiotopes on the monoclonal anti-SV40 T-Ag Ab-1 based on competitive cross-inhibition studies. One of these anti-Id preparations, designated 57B, was examined further for its in vivo modulatory capacity in mice. This anti-Id induced an Ab-3 response in BALB/c mice that recognized SV40 T-Ag (Ag+) and expressed an Id that was shared by the monoclonal anti-SV40 T-Ag Ab-1 preparation (Id+). The Id expressed on the Ab-3 differed from the Id induced in BALB/c mice immunized with the nominal SV40 T-Ag. Furthermore, characterization of the humoral immune response induced by anti-Id immunization indicated that the Ab-3 also recognized different epitopes on SV40 T-Ag when compared to the anti-SV40 T-Ag Ab-1 preparation used to generate the anti-Id. These studies indicate that monoclonal anti-Id can be used to induce humoral immune responses to a viral encoded tumor-associated Ag in vivo with 1) and Id specificity that differs from that expressed on antibodies produced by immunization with the nominal Ag and 2) an epitope specificity distinct from the Ab-1 preparation used for the production of the anti-Id.     

Idiotypic analysis of anti-I-Ak monoclonal antibodies. III. T- and B-cell responses to anti-Ia idiotopes are not modulated by syngeneic anti-idiotypic monoclonal antibodies

To investigate whether anti-idiotypic (anti-Id) antibodies activate T cells either directly or indirectly, we examined the ability of syngeneic anti-Id monoclonal antibodies (mAbs) to regulate idiotype (Id) expression, antigen-binding antibody production, and T-cell reactivity to antigen. Our idiotypic system consists of an anti-I-A mAb that carries an infrequently expressed Id. Using three syngeneic anti-Id mAbs (Ab2), we previously defined the idiotype of the 11-5.2.1.9 (11-5) anti-I-Ak mAb. Two of these mAbs, IIID1 and IA2, recognize the same or closely related epitopes on 11-5 and cross react with two additional anti-I-Ak mAbs, 8B and 39J; the third anti-Id mAb, VC6, recognizes a distinct epitope shared by 11-5 and 8B. In the present study, BALB/c (H-2d) mice were primed with varying doses of these anti-Ids and were then boosted with C3H (H-2k) spleen cells. Among 130 such primed mice, the syngeneic anti-Ids when tested at priming doses between 10 ng and 10 micrograms were unable to induce Id production. The priming anti-Id mAbs persisted in the serum of the mice and were detectable as late as 40 days after priming. Ab1 expression was not modulated in BALB/c mice immunized with KLH-coupled Ab2, however, this immunization elicited the production of Ab3 which shared idiotypes with 11-5, 8B, and 39J. BALB/c anti-C3H alloreactive T-cell clones were also not induced by anti-Id priming, nor could they be shown to bind directly to the three Ab2 used. Nevertheless, the proliferative response of one anti-I-Ak specific T-cell clone that recognizes the same epitope as 11-5, 8B, and 39J, was inhibited by the IIID1 and IA2 Ab2. Thus, a T cell can express an idiotype shared by a B cell, but the linked recognition of an Id-associated carrier determinant(s) by an alloreactive T cell is required to elicit an anti-Id antibody response. These results favor the possibility that the activation of T cells is not dependent upon their ability to bind to anti-Id, but rather on their capacity to respond to epitopes of Id-anti-Id antigen-antibody complexes formed on B cells.     

Thymus-dependent antiidiotype and anti-antiidiotype responses to a dinitrophenyl-specific monoclonal antibody

BALB/c mice were inoculated i.p. with graded doses of a DNP-specific, IgM mAb (designated 57.1). Injection with unmodified 57.1 in the absence of adjuvants resulted in the generation of an anti-Id response (Ab2) and an anti-anti-Id response (Ab3). The generation of serum anti-Id antibodies was found to be thymus dependent. Nude mice immunized with 57.1 were unable to produce a serum Ab2 response above nonimmunized controls whereas euthymic mice receiving identical doses of 57.1 produced strong Ab2 responses. To examine the specificity of serum anti-Id, sera from mice receiving 57.1 were screened against a panel of mAb representing at least five distinct VH gene families. Serum titers were significantly higher against 57.1 than against any of the other antibodies in the panel. Three of the antibodies in this panel bind FD5-1, a monoclonal anti-Id (Ab2) that also binds 57.1. However, sera from mice receiving 57.1 bound 57.1 only. Thus, the serum Ab2 response appears to be highly specific for idiotopes on 57.1. The predominant isotype of these anti-Id antibodies was IgG1. The number of isotypes detected increased in a dose dependent manner with all IgG subclasses having anti-Id specificity in sera from animals receiving the higher doses of 57.1. Further analysis of the serum demonstrated that approximately 8% of the Ab2 response was paratope-specific (inhibitable by the monovalent hapten DNP-lysine). The same sera were analyzed for the presence of Ab3 by binding to the monoclonal anti-Id antibody FD5-1. Lower serum titers of Ab3 were generated in comparison to serum titers of Ab2. Analysis of the binding specificity of the Ab3 response revealed that DNP-BSA was able to partially inhibit the binding of serum IgM and IgG Ab3 to FD5-1. A subset of the Ab3 response. Ab1' that is specific for DNP was observed in a direct binding assay where detectable amounts of DNP binding IgM, IgG1, and IgG3 isotypes were present. We have thus described a complete circuit (Ab1----Ab2----Ab3) of antibodies within the Id network by immunizing animals with an unmodified mAb in the absence of Ag or adjuvants.     

Analysis of anti-tumor antibodies in mice and rabbits induced by monoclonal anti-idiotope antibodies

Eight different mouse monoclonal anti-idiotope antibodies (mAb2) generated against a mouse monoclonal anti-human melanoma proteoglycan Ag (MPG) antibody (mAb1), MEM136, were tested for their ability to induce anti-MPG responses in mice and rabbits. All Ab2 were idiotypically cross-reactive and combining site-specific as demonstrated by competitive cross-inhibition studies and their ability to inhibit the binding of MEM136 to the melanoma cells, Colo38. However, only two Ab2, IM32 and IM06, were able to induce specific anti-TAA-specific (Ab1') responses in rabbits. When IM32 and IM06 were tested in allogeneic stains of mice for the induction of anti-MPG responses, only IM32 produced an Ab1' response. In mice, the Ab3 response induced by IM32 is idiotypically cross-reactive with its Ab1. Furthermore, the IM32-induced murine Ab3 and MEM136 recognized a similar MPG epitope on the melanoma cells because the Ab3 inhibited the binding of MEM136 to melanoma cells. The Ab3 induced by IM32 and IM06 in rabbits also recognized a similar epitope as the Ab1. In rabbits, the Ab3 response induced by IM32 and IM06 were idiotypically cross-reactive with each other. However, additional studies indicated that the majority of Ab3 induced by IM32 were IM32 Id-specific and lacked IM06 idiotopes. Further experimentation indicated that IM32-induced rabbit Ab3 were biologically active as demonstrated by the ability of the Ab3 to inhibit melanoma cell invasion in a Matrigel assay.     

An anti-idiotype vaccine elicits a specific response to N-glycolyl sialic acid residues of glycoconjugates in melanoma patients

We generated the 1E10 gamma-type anti-idiotype mAb (Ab2) specific to an Ab1 mAb able to react specifically with N-glycolyl-containing gangliosides and with Ags expressed on human melanoma and breast carcinoma cells. This Ab2 mAb induced an Ab response in animal models sharing immunochemically defined idiotopes with the Ab1. The treatment of tumor-bearing mice with 1E10 mAb induced a strong antitumor activity. A clinical trial was conducted in 20 patients with advanced malignant melanoma. Patients were treated with six intradermal injections of aluminum hydroxide-precipitated 1E10 anti-Id mAb given at 2-wk intervals. Sixteen of the 17 patients who received at least four doses of the anti-Id vaccine develop Ab3 Abs capable of inhibiting Ab2 binding to Ab1 (Ab3Id+). In contrast to the incapacity of 1E10 mAb to generate Ab3 Abs with the same antigenic specificity as the Ab1 mAb in mice, a very specific and strong Ab3 response against N-glycolyl-containing gangliosides was induced in 16 patients (Ab3Ag+). No evidence of serious or unexpected adverse effects has been observed in this clinical trial. 1E10 anti-Id vaccine was safe, well tolerated, and immunologically effective, with most patients being able to generate a specific immune response against 1E10 and Neu-glycolyl-GM(3) ganglioside.     

Collagen-Induced Arthritis Suppressed with Monoclonal Anti-Idiotypic Antibody

In foregoing work, we identified at least 5 distinct epitopes on human type II collagen (CII), using 8 murine monoclonal antibodies (mAb) against human CII, and suggested that a species-nonspecific epitope on CII recognized by anti-CII mAb termed 1-5 is an arthritogenic epitope. We also found that antibody response against a selected epitope of human CII could be induced by immunization with rabbit anti-idiotypic (Id) antibody against anti-CII mAb. The author developed and characterized monoclonal anti-Id antibodies against 1-5 mAb recognizing a putative arthritogenic epitope. The author also investigated whether the anti-Id mAb could regulate antibody response directed against a selected epitope recognized by 1-5 mAb, and the induction of collagen-induced arthritis in DBA/1J mice. DBA/1J mice intravenously preinjected with anti-Id mAb to 1-5, did not produce anti-CII antibody expressing 1-5 Id upon immunization with human CII. Furthermore, as the development of collagen-induced arthritis (CIA) in DBA/1J mice pretreated with anti-Id mAb to 1-5 was significantly suppressed, anti-Id mAb will be a useful tool for studying the regulation of antibody response to a selected epitope. This study lends support to our hypothesis that the 1-5 epitope is an arthritogenic epitope.     

Biological Mimicry of the Bluetongue Virus Core Protein VP7 by Rabbit Anti-Idiotype

A subpopulation of rabbit polyclonal anti-idiotypic antibody (anti-Id) was previously produced to a murine monoclonal antibody (mAb) (M1875) specific for the bluetongue virus core protein VP7. In this report, mimicry of VP7 by this anti-Id (designated RAb2-A) was functionally analyzed through immunization of Balb/c mice with RAb2-A or purified VP7. Animals immunized with RAb2-A were able to produce an M1875-like Ab3 antibody response with idiotype and epitope specificity resembling that of M1875 without subsequent exposure to the nominal antigen. This conclusion was supported by experiments showing that the RAb2-A-induced Ab3 antibodies (i) reacted specifically with the immunizing anti-Id; (ii) were capable of binding VP7; (iii) inhibited M1875 from binding to VP7; and (iv) inhibited M1875 from binding to RAb2-A. Similarly, mice immunized with purified VP7 also produced antibodies that exhibited characteristics such as idiotype and epitope specificity in common with M1875. No antibody response to VP7 was detected in control groups of mice immunized with either normal rabbit IgG or BHK-21 cell components. Therefore, it can be concluded that rabbit anti-Id RAb2-A mimics an M1875-defined VP7 epitope sufficiently to function as a surrogate antigen for inducing an anti-bluetongue virus response.     

Antiidiotypic antibody related to the 84 kD human sperm membrane protein

Wistar rats were inoculated with purified YWK-I antibody.The anti-idiotypic antibodies were isolated from rat sera by successive passage over affinity chromatography columns of YWK-I mAb and normal mouse Igs.Specificity of anti-Id antibody was established by ELISA.The 84kD protein inhibited the binding of anti-Id to YWK-I mAb,but failed to repress antibody against normal mouse Ig binding to YWK-I mAb.In competitive inhibition assay,84kD protein had shown the ability to compete with anti-Id binding to YWK-I mAb in a dose-dependent manner.Crude sperm extract showed a lower competitive ability.No effect was found with the irrelevant 36kD sperm protein.The antisera from the Balb/C micr immunized with AId contained Ab3 that reacted with 84kD sperm protein.The binding of anti-Id to YWK-I mAb was inhibited by Ab3 in a dose-dependent fashion and Ab3 was shown to be able to induce human sperm agglutination.These results indicate that anti-Id which may mimic an epitope of the 84kD protein could be exploited as an antigen to raise antibodies against sperm protein.     

Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine

In this study, we compared the immunogenicity and tumor-protective activity of anti-idiotypic antibodies mimicking a single tumor-associated epitope and tumor-associated antigen expressing multiple potentially immunogenic epitopes. We focused our study on the colorectal-carcinoma(CRC)-associated antigen GA733 (also known as CO17-1A/KS1-4/KSA/EpCAM). Monoclonal anti-idiotypic antibody (Ab2) BR3E4 was produced against murine anti-CRC mAb CO17-1A (Ab1) in rats. Full-length native GA733 protein was isolated from human tumor cells, and the extracellular domain protein (GA733-2E) was isolated from supernatants of recombinant baculovirus-infected insect cells by immunoafffinity chromatography. The immunomodulatory activity of the Ab2 was compared with that of the antigen, both in rabbits and in mice. Mice, like humans but not rabbits, express a GA733 antigen homologue on some of their normal tissues. Thus, these in vivo models allow the comparison of the immunogenicity of Ab2 and antigen in the presence (mice) and absence (rabbits) of normal tissue expression and immunological tolerance of the GA733 antigen homologue. In rabbits, aluminum-hydroxide(alum)-precipitated native GA733 antigen was superior to alum-precipitated Ab2 in inducing specific humoral immunity. In mice, alum-precipitated recombinant GA733-2E antigen, but not alum-precipitated Ab2, induced specific humoral immunity. However, when the Ab2 was administered to mice in Freund's complete adjuvant, specific humoral immune responses were elicited. Ab2 in complete Freund's adjuvant and GA733-2E in alum were compared for their capacity to induce antigen-specific cellular immunity in mice. Whereas lymphoproliferative responses were obtained with the recombinant antigen only, delayed-type hypersensitivity responses were obtained with both recombinant antigen and Ab2, although these responses were lower than after antigen immunization. The recombinant antigen in alum did not protect mice against challenge with antigen-positive syngeneic murine CRC cells. Similar studies with Ab2 BR3E4 mimicking the CO17-1A epitope were not possible because the tumor cells do not express this epitope after transfection with the human GA733-2 cDNA. However, similar studies with Ab2 mimicking the epitope defined by mAb GA733, which is expressed by the transfected tumor cells, indicated a lack of tumor-protective activity of this Ab2. In contrast, the full-length antigen expressed by recombinant adenovirus inhibited the growth of established tumors in mice. In conclusion, soluble antigen is a more potent modulator of humoral and cellular immune responses than Ab2, both administered in adjuvant. However, for induction of protective immunity, the immunogenicity of the antigen must be further enhanced, e.g., by expression of the antigen in a viral vector. Received: 27 December 1999 / Accepted: 27 January 2000     

Immunization with antigen bound to bispecific antibody induces antibody that is restricted in epitope specificity and contains antiidiotype.

Mice can be efficiently immunized in the absence of adjuvant using chemically cross-linked bispecific antibody (biAb) that bind to both class II MHC molecules and a protein Ag of interest. In our experiments, mice were immunized with the protein Ag hen egg lysozyme (HEL) bound to several different biAb, each of which contained a different mAb specific for a distinct (nonoverlapping) epitope of HEL. Primary and secondary serum antibody responses of the immunized mice were analyzed for their specificity for different epitopes of HEL. The results show that immunization with each HEL-biAb complex produced a bias in the epitope specificity of the primary antibody response. This bias was determined by the individual specificity of the anti-HEL mAb used in each biAb. The primary response was dominated by antibody reacting with epitopes distinct from that bound by the mAb in the immunizing complex, and was deficient in antibody that recognized the epitope bound by the biAb during immunization. This bias in antibody specificity was maintained during the secondary antibody response that followed a single challenge with soluble HEL alone. However, an additional challenge with HEL induced a switch in the specificity pattern, with increased amounts of antibody against the epitope that was previously ignored. In addition, immunization with Ag bound to biAb resulted in a substantial primary anti-Id response, detected by serum antibody specific only for the Fab'2 fragment of the mAb used in the biAb. These studies illustrate two unique features of immunization using biAb that allow for fine manipulation of the epitope specificity and anti-Id repertoires of the antibody response to whole protein Ag.     

Generation of anti-idiotypic and anti-anti-idiotypic monoclonal antibodies in the same fusion. Support of Jerne's Network Theory

Upon immunization of mice with a mAb (290A-167) directed against an epitope of Lol p I (the major allergenic determinant of Lolium perenne), both anti-idiotypic (aId) mAb (Ab2) and anti-aId mAb (Ab3) were produced. The Ab2 displayed the following internal image properties of Lol p I: it can be affinity-purified on an immobilized Id column; its binding to the anti-Lol p I mAb (290A-167) is inhibited by Lol p I; it inhibits in a dose-response fashion the binding of the specific Id to Ag. It is recognized by anti-Lol p I antisera from different species such as mouse, human, and goat. The Ab3 which binds to Lol p I was also produced from the same fusion. This binding was inhibited significantly by aId mAb (Ab2), anti-Lol p I mAb (290A-167) and Lol p I. These data indicate that the two mAb with specificity for Lol p I (290A-167 and Ab3) share similar reactivity to the Ag and that aId mAb is the internal image of the epitope recognized by the Id. We showed also that the capacity of rabbit aId Ab directed against the 290A-167 Id to inhibit the binding of Ab1 and Ab3 to Ag was almost abolished by passage over a Ab3-coated Sepharose column. This would suggest that not only are the two mAb with reactivity to Lol p I (Ab1 and Ab3) directed against identical epitopes, but that they in fact shared identical idiotopes as well. The production of identical mAb upon immunization with either the Ag or the aId mAb supports that the conceptual framework proposed by Jerne finds its biologic application in the course of an immune response.     

Activation of a functional idiotype network response by monoclonal antibody specific for a virus (M-MuLV)-induced tumor antigen

BALB/c mice were injected with IgM mAb specific for Moloney murine leukemia virus (M-MuLV)-determined cell surface Ag in an attempt to inhibit Moloney sarcoma growth. The monoclonal IgM significantly inhibited sarcoma growth when given to the mice after inoculation with Moloney murine sarcoma/leukemia virus, and also potentiated the in vivo antibody response specific for M-MuLV Ag. These responses were significantly greater than the primary response to the virus alone in age- and sex-matched control mice, and were also seen in mice which were injected with the IgM antibody only and not with virus, suggesting that an Ag-independent mechanism may be involved. The M-MuLV-specific serum antibody responses induced by the monoclonal IgM, with or without prior virus inoculation, were predominantly of the IgG1 isotype, with some IgG2a; no other isotypes were found to have titers significantly higher than in the normal response to virus alone. M-MuLV-specific IgG1 was detected only in mice injected with monoclonal IgM, and not in the response to virus alone. The same sera also had high titers of anti-idiotypic antibodies, (Ab2), as well as anti-anti-idiotypic antibodies (Ab3). It appears, therefore, that passive immunization with M-MuLV-specific IgM mAb activates an idiotypic network, which results in both Ab2 and Ab3 responses; the M-MuLV-specific response may be considered a subset of Ab3.     

Tumor idiotype vaccines. VII. Analysis and correlation of structural, idiotypic, and biologic properties of protective and nonprotective Ab2

We have previously generated and used anti-Id mAb (Ab2) to induce protective immunity against the L1210 DBA/2 tumor and for immunotherapy of established tumors. Among various anti-Id that were typed serologically as internal image Ab2 of the mouse mammary tumor virus tumor-associated Ag gp52, only one induced protective immunity and was effective in immunotherapy. In this study we compared the structural, idiotypic, and network properties of the protective and nonprotective antiidiotypic antibodies. The DNA sequence of the variable regions of six anti-Id was determined. The VH sequence of four Ab2, including the protective Ab2, are highly homologous, whereas the VL sequences differ and were assigned to different Vk families. In addition, the DH sequence region of the same four Ab2 are identical, whereas one is highly homologous and another one without homology. Search for amino acid sequence homologies between the Ab2 and gp52 showed the strongest similarities in the CDR2 of the L chain from the protective Ab2. In addition, the CDR2 region also had homology with a T cell epitope on gp52. The biologic basis of effective idiotypic mimicry was studied at the level of Ab3 induced by the Ab2. Id inhibition analysis using Ab3 induced by either protective or nonprotective Ab2, revealed differences. Thus, there is evidence for differences among the Ab1-Ab2-Ab3 cascade induced by protective and nonprotective anti-Id.     

Diversity and relative inaccuracy of internal images of antigen group A revealed by IEF analysis]

The diversity of a polyclonal anti-idiotypic response (Ab2) to a murine monoclonal anti-A (Ab1) was investigated after purification of two Ab2 populations. One was eluted from human polyclonal anti-A column and the other from Ab1. Analysis of the Ab specificity, as well as screening of the clonotypic distribution, were achieved after splitting Ab by IEF; this was followed by immunoblotting and probing with various anti-ABH mAb. The first population reacted with almost all the murine anti-ABH mAb, as well as with four human anti-A mAb, and consequently consisted of Ab2 beta. The second was composed of "true" Ab2 directed against Ab1. In the first population internal images mimicked either A Ag, or H Ag, or some epitopes common to both. This study demonstrates the plurality of internal images-bearing Ig molecules, some mimicking completely, and some only partially or even unfaithfully the nominal A determinant. The analysis of this idiotypic cascade proves the existence of a degeneracy of the initial restricted antigenic specificity. The consequences of such a process are discussed.     

Immunostimulatory CpG oligonucleotides enhance the immune response of anti-idiotype vaccine that mimics carcinoembryonic antigen

We have developed and characterized a monoclonal anti-idiotype (Id) antibody, designated 3H1, which mimics a specific epitope of carcinoembryonic antigen (CEA) and can be used as a surrogate for CEA. Anti-Id 3H1 induced anti-CEA immunity in different species of animals as well as humans and showed promise as a potential vaccine candidate in phase I/II clinical trials for colorectal cancer patients. One area of interest to us has been the development of new immune adjuvants that may augment the potency of 3H1 as a tumor vaccine. Immunostimulatory oliogonucleotides containing the unmethylated CpG motif (CpG ODN) are potent inducers of both innate and adaptive immunity and can serve as suitable vaccine adjuvants. In this study, using the CEA-transduced MC-38 murine colon carcinoma model in syngeneic C57BL/6 mice, we assessed whether a select CpG ODN (1826) can function as immune adjuvant in immunization of mice with anti-Id 3H1. Complete Freund's adjuvant (FA) was used as a gold standard in this system. A single immunization of 3H1 mixed with CpG ODN 1826 was sufficient to induce measurable anti-CEA immunity in na?ve mice. However, 3 immunizations every other week were necessary to obtain and sustain peak immune reactivity over a long period of time. With FA and 3H1, single immunization was ineffective and multiple immunizations (5 to 6) were needed to achieve and sustain peak immunity. Anti-CEA antibody reactivity was comparable in both groups, but cellular immune reactivity as measured by immune splenic lymphocyte T cell proliferation and cytoxicity assay was slightly higher in the CpG ODN group. Mice immunized with 3H1 and either CpG ODN or FA were protected from challenge by lethal doses of MC-38-CEA cells. However, the degree of protection was slightly higher and the duration of survival was somewhat longer in the group of mice treated with 3H1 plus CpG ODN. Thus, CpG ODN 1826 was faster than FA in increasing anti-tumor immunity induced by anti-Id 3H1 immunization in this prophylactic model.     

Characterization of antibodies to idiotypic determinants of monoclonal anti-sperm antibodies

Rabbit polyclonal antibodies to the idiotype of murine monoclonal anti-sperm antibodies were developed and characterized. M29.6 and M42.15 are monoclonal antibodies (mAbs) that inhibit fertilization in vivo and in vitro. Sera from rabbits inoculated with purified mAbs (Ab1) were absorbed with normal mouse and isotype-specific immunoglobulin (Ig); the anti-idiotype Ig fraction (Ab2) was isolated by protein A-chromatography or by chromatography on the corresponding idiotype column. Binding specificity of Ab2 was confirmed by measuring the reactivity of Ab2 with homologous and heterologous mAbs. Ab2 competitively inhibited 125I-labeled Ab1 binding to mouse sperm, suggesting that the Ab2 preparation possessed subpopulations directed against idiotopes similar or adjacent to the antigen-binding site of the mAb. Anti-idiotype antibodies reactive with the antigen-combining site of the anti-sperm mAb may contain subpopulations that mimic the mouse sperm epitope recognized by Ab1. Immunization with Ab2 induced anti-(anti-idiotype) antibodies (Ab3), which competitively inhibited binding of 125I-labeled Ab1 to immobilized Ab2. These results are consistent with the hypothesis that immunization of mice with antibodies to the idiotype of sperm-specific mAbs can induce antibodies that share structural similarities with the anti-sperm mAb used for their induction. Immunization with anti-idiotype antibodies that mimic sperm antigen structure represents a possible strategy for induction of immunity to sperm.     

Angiotensin II (AII)-related idiotypic network. II. Heterogeneity and fine specificity of AII internal images analyzed with monoclonal antibodies

Although the structural basis of internal images borne by beta type monoclonal anti-idiotypic antibody (Ab2) begins to be elucidated, there is little information on the repertoire of epitopes which make up the internal images expressed by polyclonal Ab2. We addressed this question by using a two-way approach in the angiotensin II (AII)-related idiotypic network, a system characterized by common occurrence of internal images on rabbit Ab2. First, two sets of internal images were purified in parallel by affinity chromatography on Sepharose 4B covalently linked to either mAb 110 (S4B-110), a mAb specific for a phenylalanine requiring carboxy terminus epitope (Phe8) on AII, or mAb 133 (S4B-133), reactive with a more central epitope also expressed on Phe8 substituted peptide analogs. The respective eluates, EL1 110 and E11 133, exhibited only partially overlapping reactivity, as demonstrated by 1) a different pattern of inhibition by various AII peptide analogues of EL1 110 and E11 133 binding to the same anti-AII antibody (Ab1) (either the homologous polyclonal Ab1 102 or mAb 133), 2) and a distinct profile of EL1 110 and EL1 133 binding to 12 biotinylated monoclonal Ab1 identifying a variety of epitopes on AII. To analyze further the respective distribution of mAb 110 and mAb 133 defined epitopes on Ab2-beta molecules, Ab2 were submitted to sequential affinity chromatography on S4B-110 followed by S4B-133, and the fractionated internal images were characterized by the pattern of binding to the various monoclonal Ab1. It was thus possible to purify two Ab2-beta subpopulations that exclusively imaged the determinant identified by mAb 110 (ii 110) or that identified by mAb 133 (ii 133). A third subpopulation which was successively retained on S4B-110 and S4B-133 expressed both internal images (ii 110 + 133), and was additionally reactive with all the other monoclonal Ab1 tested. In any case, monoclonal Ab1 binding to the different sets of internal images was totally inhibited by an excess of AII. These results indicate that the repertoire of internal epitopes is similar to that of the nominal Ag, but is scattered over distinct subpopulations of Ab2-beta molecules that can be fractionated by affinity chromatography. Some of the latter seem to bear several epitopes and resemble the whole nominal Ag, whereas others appear to image only one determinant. Second, we raised 7 anti-anti-idiotypic mAb (monoclonal Ab3) against affinity-purified Ab2-beta and analyzed their fine specificity for AII.(ABSTRACT TRUNCATED AT 400 WORDS)