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SUMMARY Baculovirus-derived recombinant simian virus 40 large tumour antigen (SV40 T-ag) was used to immunize BALB/c, C57Bl/6 and CB6/F, mice and their anti-SV40 T-ag antibody responses were examined for the ability to bind synthetic peptides representing six predicted B cell epitopes on SV40 T-ag. In C57B1/6 mice, anti-SV40 T-ag antibodies failed to bind any of the six SV40 T-ag peptides. However, the antibody responses induced in both BALB/c and CB6/F| mice recognized synthetic peptides corresponding to two distinct epitopes (amino acids 690 708 and 660 679, respectively) associated with the carboxyl-terminal half of SV40 T-ag, In addition, murine MoAbs (BALB/c) generated to native SV40 T-ag, and previously characterized as recognizing the carboxyl-terminus of SV40 T-ag by deletion mutant analysis, also bound the synthetic peptide (residues 690–708) defining the carboxyl-terminus of SV40 T-ag. These data indicate that the antibody responses induced in BALB/c and CB6/r-| mice by immunization with baculovirus-derived recombinant SV40 T-ag are capable of recognizing sequential carboxyl-terminal epitopes on SV40 T-ag defined by peptides 690 708 and 660-679. respectively. No statistically significant differences in anti-SV40 T-ag antibody titres were observed between the three inbred mouse strains. These data suggested that the fine specificities of the anti-SV40 T-ag responses as assessed by synthetic peptide binding were different in the three inbred strains of mice examined. Finally, in vivo tumour challenge studies comparing recombinant SV40 T-ag with the two carboxyl-terminus peptide epitopes indicated that some tumour immunity was induced in BALB/c, but not CB6/F1 mice, by immunization with peptide 690 708 conjugated to a carrier protein. These studies suggest that the carboxyl-terminal region of SV40 T-ag represents A continuous sequential epitope involved in both the antibody response to SV40 T-ag and tumour immunity in BALB/c mice.

Borrelia burgdorferi, an agent of Lyme disease, is killed by some monoclonal antibodies in the absence of complement or phagocytes. In the present study, the bactericidal action of monoclonal antibodies against B. burgdorferi and B. hermsii, a cause of relapsing fever, was further characterized. H6831, an antibody recognizing the OspB proteins of some B. burgdorferi strains, and H4825, an antibody specific for one serotype of B. hermsii, were purified, and Fab fragments of the antibodies were prepared. In time-kill studies, more than 99.9% of strain B31 B. burgdorferi cells were killed after 30 min of exposure to H6831 Fab fragments. The MBC of the Fab fragments was 10 micrograms/ml. Electron microscopy revealed that the bactericidal Fab fragments produced numerous blebs and cell lysis of the borrelias for which they were specific. To identify the epitope for H6831, the OspB sequences of H6831-susceptible and -resistant strains and mutants were determined. The deduced OspB proteins of all H6831-resistant strains and mutants differed from the strain B31 OspB at residue 253. Murine antisera raised against a 21-mer synthetic peptide representing the region around residue 253 were specific for strain B31 by Western blot (immunoblot) and growth inhibition assays. Furthermore, the antipeptide serum inhibited the binding of H6831 to whole borrelias. These findings indicated that the linear component of the bactericidal antibody's epitope was located at or near residue 253.

The reversible denaturant-induced unfolding of immunoglobulin molecules has been analyzed by transverse urea gradient gel electrophoresis and the effects that urea-induced unfolding exerts on the functional properties associated with their variable region, i.e. antigen binding and idiotypic expression, have been determined by Western blot analysis. Results obtained from these experiments indicate that urea-induced unfolding of the immunoglobulin molecule is a highly cooperative reversible process that occurs through a two-state transition with no accumulation of intermediates. The unfolding transition has its midpoint at about 6.5 M urea and appears to be slow on the time scale of electrophoresis. Folding intermediates in rapid equilibrium with the unfolded state as well as molecular forms with different electrophoretic mobility can be detected during refolding reactions. Results from Western blot analysis confirm the highly cooperative reversible urea-induced unfolding of immunoglobulin molecules and demonstrate that the unfolding transition leads to disappearance of both antigen binding and idiotypic expression, whereas the ability to interact with antibodies directed to continuous epitopes of the variable region is preserved. After progressive removal of the denaturing agent, the variable region refolds into structures that regain the functional properties of the native conformation.

We compared the humoral immune responses induced in BALB/c mice by immunization with recombinant SV40 large tumor antigen (T-ag) with those induced by a monoclonal anti-idiotype (anti-Id), designated 58D, that is specific for SV40 T-ag-induced Id network components. We also challenged immunized mice with a lethal dose of SV40-transformed cells to assess in vivo tumor immunity. Two biweekly immunization with either SV40 T-ag or anti-Id 58D induced humoral responses that recognized both SV40 T-ag and anti-Id 58D. Four biweekly immunizations with SV40 T-ag increased the antigen-specific antibody titers and decreased the response to anti-Id 58D, while four biweekly immunizations of anti-Id 58D increased antibody titers to both itself and SV40 T-ag. Comparison of specific T-ag epitope and idiotope specificities indicated that SV40 T-ag and anti-Id 58D immunization generated responses that recognized a similar epitope on SV40 T-ag and expressed a shared idiotope recognized by anti-Id 58D. SV40 T-ag immunized mice challenged with a lethal dose of SV40-transformed cells were completely protected and no tumors were observed. This is despite the fact that little or no SV40 T-ag-specific cytotoxic T-lymphocyte activity was detectable. In contrast, only 3 of 10 mice immunized with anti-Id 58D were protected from a lethal challenge. These results indicate that, although monoclonal anti-Id immunization can induce responses that recognize similar SV40 T-ag epitopes and express shared idiotopes associated with antibodies to SV40 T-ag, the recombinant antigen itself induces superior in vivo tumor immunity.

We have characterized a series of mouse monoclonal anti-CD4 and describe both their CD4 epitope recognition and Id expression. We also determined the V region gene sequences of these antibodies in an attempt to correlate epitope recognition and Id expression with V region sequence. All of these preparations recognize epitopes that cluster around the HIV gp120 binding site on the human CD4 molecule. However, we observed differences in epitope recognition among the anti-CD4 preparations, based on either competitive inhibition assays or functional assays, such as syncytium inhibition. Analysis of Id specificities using a polyclonal anti-Id generated against anti-Leu 3a indicated that five of the seven monoclonal anti-CD4 expressed a shared Id. Based on V region gene sequences, the V region kappa-chain (V[kappa]) from each of the seven antibodies was encoded by the V[kappa]21 gene family and expressed the J[kappa]4 gene segment. Those preparations that expressed the shared Id with anti-Leu 3a have virtually identical V[kappa] sequences, with a high degree of homology in the CDR. The VH region gene sequences of six of the seven antibodies also shared overall homology and appeared to be encoded by the J558 VH gene family. The seventh anti-CD4 VH region is encoded for by the VHGAM gene family. The majority of these antibodies used JH3 gene segment, although the JH2 and JH4 gene segments were also represented. In addition, several of these antibodies share a common sequence organization within their V-D-J joining regions that appears to involve N and P sequences to generate unique D segments. Together, these data suggest that differences in epitope recognition among the monoclonal anti-CD4 may reflect sequence variability primarily within the CDR3 region of both V[kappa] and VH. The basis for the detection of a shared Id most likely reflects the high degree of homology within the V[kappa] region sequences. In addition, these data, which are based on a limited analysis, suggest the possible restricted use of V region germ-line gene families in the secondary antibody response of BALB/c mice to specific epitopes on the human CD4 molecule.

A panel of mouse monoclonal anti-CD4 antibodies was characterized in terms of idiotypic expression by using specific anti-idiotypic antibody (anti-Id) reagents generated in rabbits immunized with anti-Leu3a, a monoclonal anti-CD4 which inhibits the human immunodeficiency virus (HIV) gp120 binding to CD4. Direct binding and competitive inhibition assays demonstrate that the majority of monoclonal anti-CD4 antibodies able to recognize CD4 epitopes overlapping the epitope recognized by anti-Leu3a expressed an antigen-combining site-related cross-reactive idiotype (IdX). Western blot analysis was used to demonstrate that this IdX is associated primarily with the light (L) chain of the monoclonal anti-CD4 antibodies. To further characterize the structural basis of the IdX, the nucleotide sequence of the variable region of the L kappa chain of anti-Leu3a was determined. Peptides corresponding to the first, second, and third complementarity determining regions (CDRs) of the L chain of anti-Leu3a were synthesized and used to immunize rabbits. All anti-peptide antisera recognized the immunizing peptide, the cognate anti-Leu3a molecule, and several other monoclonal anti-CD4 antibodies by direct binding assays. Western blot analysis utilizing the anti-CDR peptide reagents demonstrates that the reactivity to the monoclonal anti-CD4 antibodies was L chain-specific. The anti-Id generated by immunizing with the intact anti-Leu3a molecule failed to recognize the three L chain-derived CDR synthetic peptides, suggesting that the IdX requires the presence of the three-dimensional configuration of the L chain for its expression. The broad range of reactivity exhibited by the antipeptide antisera indicates that the majority of mouse monoclonal anti-CD4 antibodies characterized in this study utilize L chains encoded by a single germ line variable (V) region kappa (V kappa) chain gene or by V kappa genes that belong to the same gene family.

A series of mouse monoclonal anti-CD4 preparations was characterized for the ability to recognize overlapping epitopes on CD4 and to inhibit HIV/simian immunodeficiency virus (SIV) syncytium formation. Based on this characterization, mAb able to recognize CD4 epitopes overlapping the HIV binding site were selected and used to immunize nonhuman primates to elicit the production of specific anti-Id antibodies. Five baboons and five rhesus monkeys were immunized with either individual or a cocktail consisting of several monoclonal anti-CD4 preparations. All the nonhuman primates produced specific anti-Id that recognized either private or cross-reactive Id depending on the monoclonal anti-CD4 used to generate the anti-Id response. Inhibition assays were performed to ascertain the ability of: 1) soluble CD4 to inhibit the Id-anti-Id reaction and 2) the various anti-Id to inhibit the CD4-monoclonal anti-CD4 reaction. These studies demonstrated that some of the anti-Id recognized a cross-reactive Id that was associated with the Ag-combining site. In addition, some of the anti-Id weakly recognized SIV gp120 by Western blot analysis. These studies may be useful in designing experiments that may lead to a better understanding of the CD4-HIV gp120 interaction and to the production of Id and/or anti-Id reagents that might be used to manipulate this virus-receptor interaction.

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.

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.

Serum neutralizing antibodies against the human immunodeficiency virus were frequently detected in infected individuals, and low or absent serum neutralizing titers correlated with poor prognosis. Multiple diverse human immunodeficiency virus isolates were found to exhibit similar susceptibility to neutralization by a panel of human seropositive sera, suggesting that neutralizing antibodies are largely directed against conserved viral domains. Furthermore, utilizing antisera raised against a library of synthetic env peptides, four regions which are important in the neutralization process have been identified within both human immunodeficiency virus envelope glycoproteins (gp41 and gp120). Three of these are in conserved domains and should be considered for inclusion in a candidate vaccine.

A synthetic hepatitis B surface antigen (HBsAg) peptide was prepared containing amino acid residues 122-137 of the major HBsAg polypeptide. This peptide was cyclized by the introduction of an intrachain disulfide bond between cysteine residues at positions 124 and 137 because previous studies had shown that intact disulfide bonds are critical for maintenance of HBsAg activity. An anti-HBs response was produced in mice by free peptide entrapped in liposomes. However, the immunogenicity was enhanced by aggregation into micelles, and by coupling to tetanus toxoid. Analysis of the peptide with a panel of monoclonal antibodies showed that peptide 122-137 contained a conformation (discontinuous) group a epitope and a sequential (continuous) subgroup y epitope. In addition, the cyclic peptide inhibited a human anti-HBs idiotype-anti-idiotype reaction with specificity for group a determinant(s). The potential for synthetic peptides for hepatitis B virus vaccine development is discussed.

A polyamide-based solid-phase support containing an acid-stable p-(oxymethyl)benzoic acid handle to anchor the COOH-terminal amino acid was utilized in the production of synthetic peptides analogous to amino acid sequences 503-532 from the human immunodeficiency virus (HIV) envelope glycoprotein. The resin-bound peptide was used to induce an antibody response to the native form of glycoprotein 120 in both rabbits and mice. This epitope was detected on the surface of HIV-infected cells and was capable of inducing an in vitro neutralizing HIV antibody response. In addition, sera from some individuals exposed to HIV react with this peptide bound to the resin in a solid-phase immunoassay. These data indicate that we have identified a neutralizing antigenic determinant present on the amino-terminal glycoprotein 120 subunits of HIV by utilizing resin-bound synthetic peptides.

Two IgM monoclonal antibodies (McAb) that recognized distinct antigenic determinants of herpes simplex virus type 2 (HSV2) were shown to share idiotypes. Seven other IgM McAb generated to either HSV2 or the major envelope glycoprotein of HSV2, VP119, did not express this shared idiotype. The idiotypic determinants were associated with the antibody-combining site, because HSV2 inhibited the idiotype-anti-idiotype reactions. Anti-HSV2 sera raised in BALB/c mice also expressed the idiotype shared by the two anti-HSV2 McAb.

Anti-Id antisera generated to purified anti-HBs isolated from two different individuals recognized a common idiotype. This idiotype was detected in purified anti-Hbs preparations from three individuals and also in anti-HBs-positive sera obtained from six hemophilic patients. The ability of both HBsAg and a virus-derived HBsAg native polypeptide to inhibit the idiotype-anti-idiotype reaction suggested that the anti-HBs idiotype was associated with the antibody-combining site.

Anti-idiotype antibodies to a mouse monoclonal antibody A-12 directed against HBsAg were produced in rabbits. The anti-Id consisted of an Ab-2 alpha preparation that did not display any detectable internal image activity. Immunization of BALB/c mice with the anti-Id (Ab-2 alpha) coupled to KLH induced an anti-HBs response without subsequent HBsAg exposure. No anti-HBs was detected in control groups of mice immunized with other rabbit anti-Id-KLH preparations. The anti-HBs containing sera from mice immunized with the Ab-2 alpha were able to inhibit the Id-anti-Id reaction, indicating that an Id-positive, anti-HBs response was induced. This idiotype is not normally expressed during the murine immune response to HBsAg and suggests that noninternal image anti-Id activates silent clones. This study, along with our previous results obtained with the use of internal image anti-Id, suggests that there is more than one Id network operational during the BALB/c murine immune response to HBsAg.

Anti-idiotype antibodies that recognize a common human idiotype present on antibodies to hepatitis B surface antigen (anti-HBs) from individuals naturally infected by hepatitis B virus (HBV) and an interspecies idiotype on anti-HBs produced by active immunization with hepatitis B surface antigen (HBsAg) were used in vivo to modulate idiotype networks in mice. Injection of anti-idiotype antibodies without subsequent HBsAg stimulation induced an anti-HBs response. The anti-idiotype-induced anti-HBs recognized the group-specific a determinant (s) of HBsAg, which has been shown to be responsible for induction of protective immunity to HBV in both chimpanzees and humans. In addition, the anti-HBs expressed an interspecies idiotype which is shared by humans naturally infected with HBV. Prior inoculation of mice with alum-adsorbed anti-idiotype potentiated the immune response to a subsequent inoculation with either native HBsAg particles or with a synthetic HBsAg peptide. These data suggest the potential use of anti-idiotype antibodies as a vaccine or vaccine potentiator for HBV.

A synthetic peptide (SP1), corresponding to the amino acid residues 122 through 137 of the major polypeptide derived from hepatitis B surface antigen (HBsAg), subtype ayw, was analyzed for the presence of the major epitopes of HBsAg. Both a cyclic form, produced by introduction of an intrachain disulfide bond, and a linear form of the peptide were characterized. A panel of monoclonal antibodies with defined specificity for the cross-reactive group a antigenic determinant(s) and for the y and w subtype specificities was used for this analysis. The cyclic, but not the linear, form of SP1 reacted with five of 14 anti-a monoclonal antibodies, demonstrating that the cyclic peptide contains a conformation-dependent a epitope. Only one anti-a antibody was found to react with both cyclic and linear forms of SP1. Because SP1 failed to react with the remaining 8 anti-a monoclonal antibodies, it was concluded that the a antigenic reactivity associated with HBsAg contains an additional epitope(s) unrelated to that expressed on SP1. Both cyclic and linear SP1 reacted with three of three anti-y monoclonal antibodies, indicating that a sequential y epitope is also present on SP1; no w reactivity was detected. Analysis of the idiotypes associated with the monoclonal antibodies showed those that combined with cyclic SP1 also inhibited the binding of a common human anti-HBs (CHBs) idiotype with its rabbit anti-idiotype serum, whereas a monoclonal antibody that did not react with the cyclic SP1 epitope failed to inhibit the CHBs idiotype-anti-idiotype reaction. Thus, the conformational a epitope present on cyclic SP1 appears to contain the predominant epitope recognized by humans in response to a natural HBV infection.