Interstrain cross-reactive idiotypes on monoclonal antibodies to an encephalitogenic myelin basic protein peptide.

In order to assess the role of idiotype (Id) and the anti-Id network in murine experimental autoimmune encephalomyelitis (EAE), Id-bearing monoclonal antibodies (mAb) to human myelin basic protein (MBP) peptide acetyl 1-9, as well as mAb anti-Id, were developed in EAE-susceptible PL/J mice (H-2u). These mice recognize MBP residues acetyl 1-9 as an encephalitogenic determinant. Reactivities of PL/J Id-bearing mAbs to MBP and to MBP peptides were identical to those of mAbs generated against the same MBP peptide in EAE-resistant BALB/c mice (H-2d), even though isotypes of the mAbs differed. By using an inhibitory ELISA and immunoblotting, it was demonstrated that one PL/J mAb anti-Id recognized a public or framework Id, whereas another PL/J mAb-anti Id was directed to a private Id more restricted to the paratopic site. Two Id-bearing PL/J mAbs shared a cross-reactive Id (IdX) on the light chain, and an interstrain IdX was present on both the heavy and light chains of mAbs raised in PL/J and BALB/c mice to the same MBP peptide. The PL/J mAb anti-Id was capable of cross-regulating the production of Id-bearing mAbs by hybridomas across murine strains. These findings suggest that a restrictive family of germ-line genes encode for these Id-bearing antibodies to MBP peptide, irrespective of whether the MBP peptide is encephalitogenic in the murine strain immunized. Manipulation of the Id network may provide a means for modifying autoimmune demyelinating diseases of the central nervous system.     

Analysis of the Repertoire of Anti-Idiotypic B-Cell Responses to Self-I-Ak- or -I-Ek-Reactive Monoclonal Antibodies in A.TL Mice

Twenty anti-idiotypic antisera (anti-Ids) were produced in A.TL mice to self-I-Ak or -I-Ek-reactive monoclonal antibodies (mAbs), constructed in the A.TH anti-A.TL combination. The reactivity of these anti-Ids was examined in a panel of 31 anti-Iak A.TH mAbs, using direct idiotype binding, cross-competitive inhibition of idiotype binding, and isoelectrofocusing (IEF) assays. Among 13 anti-Ids produced against anti-I-Ak mAbs, one only recognized individual idiotypic specificities (IdIs) on its corresponding mAb, while the 12 others identified homologous IdIs and recurrent idiotypic specificities also expressed on heterologous anti-I-Ak and/or I-Ek mAbs. Two sets of major cross-reactive idiotypes (IdXs) were characterized on two groups of mAbs recognizing public Ia.1, I-Ak,f,u and r) or private (Ia.2, I-Ak) determinants clustered in two spatially distinct epitope regions of the I-Ak molecule, respectively. By contrast, most (5/7) of the anti-Ids raised against mAbs recognizing polymorphic or monomorphic (Ia.7-like) I-Ek determinants displayed specificity apparently restricted to their corresponding mAb IdIs. This finding contrasted with the previous characterization, using xenogeneic anti-idiotypic reagents, of an interstrain IdX expressed on all mAbs defining Ia.7-like determinants in the IEk epitope group I. These data indicate that A.TL mice can readily develop anti-idiotypic responses towards self Ia-reactive mAb minor idiotypes (IdIs) and that recognition of anti-Iak mAb IdXs in such mice is preferentially observed when anti-I-Ak mAbs are used as immunogens.     

Anti-cyclosporine monoclonal antibodies and their anti-idiotopic counterpart: structure and biological activity.

In order to study the structural and functional mimicry of an antigen by anti-idiotypic antibodies, we generated anti-idiotopic monoclonal antibodies (anti-Id mAbs) against a mAb (R45-45-11) with specificity for the immunosuppressive cyclic undecapeptide cyclosporine (Cs; Sandimmune). Three out of five anti-Id mAbs inhibited the binding of Cs to the anti-Cs mAb R45-45-11. All anti-Id mAbs cross-reacted only with one (anti-Cs mAb V45-271-10) out of 19 anti-Cs mAbs. The anti-Cs mAb V45-271-10 recognizes an epitope on the Cs molecule which is very similar to that recognized by R45-45-11. R45-45-11 and V45-271-10 differ only by one amino acid in the variable region. The anti-Id mAbs which recognize combining site-associated idiotopes (Ids) reverse the blocking effect of the anti-Cs mAb R45-45-11 on Cs immunosuppression in vitro. The sequences of the variable regions of heavy and light chain of one anti-Id mAb were determined. X-ray analysis of the corresponding Fab fragment, either alone or complexed with the Fab fragment of the Id, is currently in progress.     

Selection of Phage-displayed Peptides Recognised by Monoclonal Antibodies Directed against the Lipopolysaccharide of Brucella

Panning and screening of various phage display libraries with monoclonal antibodies (mAbs) directed against the O-chain of the lipopolysaccharide (LPS) of Brucella sp. allowed the identification of peptidic mimotopes of some O-chain epitopes. Four mAbs were tested. The A76–12G12 mAb, which is specific for LPS of all strains of Brucella, either A-or M-dominant, did not yield any peptidic mimotope, despite a specific yield enrichment during the rounds of panning. The B66–4F9 mAb, that recognises an epitope common to both Brucella sp. and Yersinia enterocilitca O:9 strains, allowed the selection of only one phage clone that was shown to be an antigenic but not immunogenic mimotope. The B66–2C8 and A15–6B3 mAbs, respectively, specific for the LPS of A-dominant and M-dominant Brucella sp., yielded several sequences, which allowed the determination of consensus sequences. These consensus will be of high interest for the construction of second generation libraries. For the best binding peptides, competition with LPS for the binding to the mAb is detected, which suggests that the peptides bind to the paratope of the mAb. The phages selected from the libraries were used to immunise mice, and a weak antibody response directed against LPS has been observed for some peptides. These data suggest that a subset of the selected peptides are immunogenic mimotopes of the LPS epitopes.     

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.     

Epitope analysis of an immunodominant domain on the P1 protein of Haemophilus influenzae type b using synthetic peptides and anti-idiotypic antibodies.

Synthetic peptides, anti-idiotypic antibodies (anti-Id) and human and murine monoclonal antibodies (mAbs) were used to further define a major antigenic domain on the outer membrane P1 protein (OMP) of Haemophilus influenzae type b (Hib). Synthetic peptides were elaborated from the known primary sequences of the P1 protein of prototype Hib strains MinnA (OMP subtype 1H) and 8358 (OMP subtype 6U). By peptide mapping, antibodies are categorized into three groups: A, B and C. A first epitope on the P1 from strain MinnA was identified by the reactivity of one set of murine anti-P1 mAbs with the two overlapping peptides 11H and 13H, corresponding to amino acid residues 384-412 and 400-437, respectively. On the basis of their reactivity with both peptides, these mAbs were designated as group A. Anti-Id obtained from mice immunized with two group A mAbs reacted specifically with all group A mAbs. A second epitope on the same P1 protein was identified by the reactivity of the peptide 13H with another distinct set of murine anti-P1 mAbs assigned to group B. This group of mAbs did not recognize the peptide 11H. Murine anti-Id which were prepared against one group B mAb inhibited the attachment of this mAb to outer membrane preparations, whereas the binding of the other group B mAbs was not affected, suggesting that these mAbs represent a heterologous group of mAbs. The epitope(s) recognized by two human anti-P1 mAbs was (were) distinct from the ones recognized by murine mAbs since no reactivity with the peptides was observed. Similarly, the binding of the two human mAbs to the P1 antigen was not inhibited by anti-Id raised against group A or B mAbs. Interestingly, an epitope on a different P1 protein recovered from strain 8358 was identified by the reactivity of group C murine mAbs with the peptide 13U, which occupies the same position on the P1 protein as 13H but differs from the latter by 10 amino acid residues. Our studies demonstrated the presence of several distinct surface-exposed B-cell epitopes within the antigenic domain which was defined previously on the P1 protein of Hib MinnA. Furthermore, we showed the immunodominance of this region on two different P1 proteins. None of the mAbs, however, had a bacteriolytic or protective activity against Hib strains. We suggest that the surface-exposed immunodominant region on the OMP P1 of Hib do not induce protective antibodies against Hib infection.     

抗前列腺特异性膜抗原膜外区多肽单克隆抗体的研制及初步应用

目的建立前列腺特异性膜抗原（PSMA）膜外区多肽杂交瘤细胞株，并对其分泌的PSMA单克隆抗体进行初步鉴定，为PSMA的功能研究和人源化抗体的制备奠定基础，以求进一步用于前列腺癌的诊断和治疗。方法使用人工合成多肽免疫BABL／c小鼠，采用PEG融合技术建立杂交瘤细胞株，制备单克隆抗体。通过免疫荧光法、酶联免疫吸附法及斑点金标法确定单克隆抗体的交叉反应性、亲和力及免疫球蛋白的类型和亚类。结果获得两株可稳定分泌PSMA单克隆抗体的杂交瘤细胞，4F4为IsG1类，1F1为IgC3类。两株单抗均能识别LNCap细胞表达的PSMA蛋白，与不表达PSMA的PC-3、SP2／0等细胞无交叉反应。杂交瘤细胞株1F1培养上清效价为1：40。腹水效价为1：6400；而杂交瘤细胞株4F4培养上清效价为1：80。腹水效价为1：8000。结论成功地制备出两株抗PSMA单克隆抗体，均具有良好的特异性和亲和力，为进一步建立免疫分析方法。进行PSMA相关研究奠定了基础。     

Immune response to bovine viral diarrhea virus induced by anti-idiotypic antibodies.

We have previously prepared rabbit anti-idiotypic antibodies (anti-Ids) against the neutralizing monoclonal antibodies (MAbs) specific for the gp53 of bovine viral diarrhea virus (BVDV). The anti-Ids, purified by sequential immunoaffinity chromatography, inhibited the immunizing MAb from binding to the original antigens and blocked BVDV infection of cell cultures. This study evaluated immune responses in mice to the purified anti-Id reagents. BVDV-specific neutralizing antibodies were induced by the anti-Ids. The antisera (Ab3) induced by the anti-Ids immunoprecipitated gp53 from BVDV-infected Madin-Darby bovine kidney (MDBK) cell lysates. However, lymphocyte-proliferative responses were specific only for the respective immunizing antigens. These results suggest that the anti-Ids may bear an internal image of the gp53 to stimulate production of antibody but not to stimulate a virus-specific cellular immune response in mice.     

Protection Against a Syngeneic Mammary Adenocarcinoma by Administration of Idiotypic and Anti-Idiotypic Antibodies

A hybridoma secreting a monoclonal antibody (mAb) with specificity for tumor-associated cell surface antigens of a transplantable murine mammary adenocarcinoma (SMC-168) was prepared by fusion of syngeneic C3H/He spleen cells with SP2 myeloma cells. Mice which were pretreated with this mAb (C–73) were significantly resistant to the outgrowth of a tumorigenic dose of SMC-168 cells when compared to controls. The treated nice developed tumor-specific cell-mediated immunity, measured by leukocyte adherence inhibition (LAI), which was equal to that of mice immunized with live tumor cells. The IgG fraction from serum of mice receiving mAb C-73 contained antibodies which would bind to that mAb suggesting the presence of anti-idiotypic antibodies (anti-Id). This binding could be partially inhibited by a soluble l-butanol cell surface extract of SMC-168. Rabbits were immunized with mAb C-73 to produce a polyclonal anti-Id. The purified and absorbed IgG fraction of this serum would bind only to mAb C-73 and not to other mAbs of the same isotype or normal C3H/HeN IgG. Binding of the rabbit anti-Id to mAb C-73 could be partially inhibited by soluble tumor-associated antigen extracted from SMC-168. Mice immunized with this polyclonal anti-Id vaccine developed tumor-specific cell-mediated immunity and were significantly resistant to the outgrowth of a tumorigenic dose of SMC-168.     

Protection Against a Syngeneic Mammary Adenocarcinoma by Administration of Idiotypic and Anti-Idiotypic Antibodies

A hybridoma secreting a monoclonal antibody (mAb) with specificity for tumor-associated cell surface antigens of a transplantable murine mammary adenocarcinoma (SMC-168) was prepared by fusion of syngeneic C3H/He spleen cells with SP2 myeloma cells. Mice which were pretreated with this mAb (C-73) were significantly resistant to the outgrowth of a tumorigenic dose of SMC-168 cells when compared to controls. The treated nice developed tumor-specific cell-mediated immunity, measured by leukocyte adherence inhibition (LAI), which was equal to that of mice immunized with live tumor cells. The IgG fraction from serum of mice receiving mAb C-73 contained antibodies which would bind to that mAb suggesting the presence of anti-idiotypic antibodies (anti-Id). This binding could be partially inhibited by a soluble l-butanol cell surface extract of SMC-168. Rabbits were immunized with mAb C-73 to produce a polyclonal anti-Id. The purified and absorbed IgG fraction of this serum would bind only to mAb C-73 and not to other mAbs of the same isotype or normal C3H/HeN IgG. Binding of the rabbit anti-Id to mAb C-73 could be partially inhibited by soluble tumor-associated antigen extracted from SMC-168. Mice immunized with this polyclonal anti-Id vaccine developed tumor-specific cell-mediated immunity and were significantly resistant to the outgrowth of a tumorigenic dose of SMC-168.     

Anti-idiotypic antibodies to feline leukemia virus: an approach for retroviral immunization strategies.

The present study describes an approach to the development and use of anti-idiotypic antibodies as a possible immunization strategy to prevent retroviral infection. The rationale for using anti-idiotypes (anti-Ids) to try to elicit an antigenic-specific immune response is examined, and the production and characterization of polyclonal and monoclonal anti-Ids are described. Several techniques were used to determine antigenic mimicry and anti-Id subtypes. The potential use of anti-Ids in feline leukemia virus (FeLV) receptor studies and vaccine trials in vivo were investigated. Results from these studies suggest that the anti-Id strategy is feasible for the FeLV model. Polyclonal Ab2 reagents were developed that blocked virus-receptor binding and thus inhibited viral infection in vitro and induced humoral immune responses in 6- to 8-week old kittens characterized by production of Ab3 with the ability to bind the original FeLV envelope protein gp70 as assessed by Western blot analysis.     

Idiotypic determinants on monoclonal autoantibodies to the Thy-1 antigen.

Distinct monoclonal autoantibodies directed against the Thy-1 antigen differ in their reactivity with various target cells. The aim of the present study was to analyse the idiotypic specificities present on various monoclonal autoreactive Thy-1 antibodies. Xenogeneic anti-idiotypic (anti-Id) reagents were prepared by immunizing rabbits either with the 20-10-5 or with the C16-31 monoclonal Thy-1 autoantibodies. Both of the anti-Id reagents reacted with the immunizing MoAb in ELISA and inhibited the interaction of the immunizing MoAb with Thy-1-positive target cells. The anti-20-10-5 reagent reacted in ELISA inhibition tests with the C16-31 MoAb, yet failed to inhibit the binding of C16-31 MoAb to thymocytes. Anti-C16-31 Id reacted with the 20-10-5 MoAb both in ELISA assays and in cell-bound fluorescence inhibition tests. These results indicate that anti-C16-31 Id detects an Id determinant shared by C16-31 and 20-10-5 MoAb, which is a binding site-related idiotype, while anti-20-10-5 Id detects an idiotypic determinant shared by these two MoAb that is not located at the combining site of C16-31 MoAb. In contrast to the marked cross-reactivity found between C16-31 and 20-10-5 MoAb, the two anti-Id reagents showed only a weak cross-reactivity with five other Thy-1 autoantibodies tested, including those autoantibodies that showed patterns of reactivity similar to those of C16-31 and 20-10-5, with various target cells. It is concluded, therefore, that individual autoreactive Thy-1 MoAb possess distinctive idiotypic determinants, although some cross-reactivity can be detected between certain MoAbs.     

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.     

Monoclonal Antibodies against Measles Virus Haemagglutinin React with Synthetic Peptides

The ability of 17 monoclonal antibodies (MoAb) against measles virus haemagglutinin (MV-H) to bind to 10 selected MV-H-specific synthetic peptides was tested in an enzyme immunoassay (EIA). Three peptides representing residues 126-135 (close to the NH2 terminus), 309-318 (middle), and 587-596 (C-terminal) reacted with MoAb designated 48, I29, and 18, respectively. Binding of MoAb I29 to purified virus was abolished after pre-incubation with the peptide 309-318. Similarly, MoAb 48 did not bind to the virus after absorption with the peptide 126-135. Longer peptides of 19 residues from the regions reacting with the MoAb were also synthesized and tested in EIA. None of the MoAb recognized these longer peptides when the latter were bound as free peptides on solid phase. However, MoAb I29 binding to purified virus was blocked equally well by peptides 304-322 and 309-318. In contrast, peptide 121-139 absorbed the reactivity of the MoAb 48 much more weakly than the shorter peptide 126-135, suggesting that the conformation of the longer peptide in solution is different. To analyse affinities in the antigen-antibody reactions, the plates were washed with buffers of varying pH after absorption of the MoAb to MV or peptides. The MoAb I29 bound both to MV and peptide 309-318 with equal affinity, but MoAb 48 and 18 bound to the peptides 126-135 and 587-596 with lower affinity than to the virus. This study indicates that regions corresponding to amino acids 126-135, 309-318, and 587-596 define antigenic sites of the H protein.     

Spreading of the immune response to different myelin basic protein peptides in chronic experimental autoimmune encephalomyelitis in B10.RIII mice

B10.RIII mice develop chronic and relapsing experimental autoimmune encephalomyelitis (EAE) after immunization with the myelin basic protein (MBP) peptide 89-101 (VHFFKNIVTPRTP). To investigate the basis for the chronicity of the disease, the subsequent development of an immune responses to other parts of the MBP protein were investigated. Onset of disease occurs 9-25 days after immunization with MBP89-101. T cell responses towards a series of MBP peptides were assessed in an enzyme-linked immunospot assay detecting single cells secreting IFN-γ. There were responses not only to MBP89-101, but also towards peptides derived from sequences outside of MBP89-101. These peptides were of two kinds: those with sequences completely outside the 89-101 stretch of MBP; and those sharing a short sequence with MBP89-101 depending on alternative splicing of MBP mRNA. Immunization with these peptides also produced chronic EAE and a spreading of the immune response to other MBP peptides. Immunization with stepped peptides around the relevant region (MBP87-110) showed that peptides sharing a 6-amino-acid motif induced EAE after immunization. After MBP 89-101 peptide immunization, T cells isolated from lymph nodes did not cross-react in vitro to the other peptides sharing this motif. We suggest that one mechanism for the development of relapses during the disease course is the recruitment of new T cells with specificity for MBP peptides not derived from the peptide used for immunization. This is the first time such a mechanism has been demonstrated in a chronic autoimmune disease model.     

Peptide-based analysis of amino acid sequences important to the biological activity of eosinophil granule major basic protein

Synthetic peptides corresponding to amino acid sequences in eosinophil granule major basic protein (MBP) were evaluated for cytotoxic activity toward K562 cells and for ability to stimulate basophil mediator release. Results obtained using 14 peptides spanning the 117-amino acid sequence of MBP in overlapping fashion indicated that the activities mapped to peptide sequences near the amino and carboxy termini of MBP. The activity of these regions was confirmed using two peptides corresponding to MBP residues 18-45 and 89-117. A 20-h incubation with 5 microM peptide 18-45 or peptide 89-117 caused approximately the same levels (>60%) of cytotoxicity in K562 cells as 5 microM MBP. Similarly, a 30-min incubation with peptides 18-44 and 89-117 stimulated basophil histamine release in a concentration-dependent manner over the range of 5-20 microM. The level of release stimulated by 20 microM peptide 89-117 approached that stimulated by 2 microM MBP. A 20 microM concentration of peptide 89-117 also stimulated leukotriene C4 (LTC4) production by the basophils. Neither peptide 18-45 nor peptide 89-117 was cytotoxic for basophils under the experimental conditions for histamine and LTC4 release, as determined by 51Cr release. These results indicate that two MBP peptide sequences, including one (89-117) that contains a unique carbohydrate-binding region, share the biologic activities of MBP.     

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.     

DNA and Heparin Alter the Internalization Process of Anti-DNA Monoclonal Antibodies According to Patterns Typical of Both the Charged Molecule and the Antibody

The internalization into CHO-K1 fibroblasts of three polyreactive monoclonal IgG2a anti-DNA autoantibodies (mAbs), F14.6, J20.8 and F4.1, isolated from the same unimmunized (NZBxNZW) F1 mouse, and synthetic peptides derived from F4.1 was studied using a technique which quantifies nuclear accumulation. The localization of the mAbs was intranuclear. We compared the influence of two negatively-charged molecules, DNA or heparin. At low concentrations, DNA had dual effects-inhibitory or stimulatory-depending on the mAb. Heparin was inhibitory or had no effect. The possibility that proteoglycans are 'receptors' recognized by anti-DNA mAbs which bind through heparin-sensitive reactions, was explored. Only F4.1 internalization was partly inhibited in glycosaminoglycan-deficient cells. We propose that the complex alterations of internalization patterns of these polyreactive mAbs by the two negatively charged molecules can be explained by (a) the potential of polyreactive mAbs to bind to various charge (or conformation-) dependent 'receptors', (b) the potential of a subclass of mAbs complexed with DNA to utilize additional 'receptor(s)'. Glycosaminoglycans were required for internalization of F4.1-derived peptides, which remained extranuclear, suggesting that nuclear internalization of mAb F4.1 is a multistep process that requires certain sequences present on the intact mAb.     

Characterization of a dominant anti-Ia idiotype using the IA mutant mouse strain B6.C-H-2bm12

Initial studies of antibody recognition of Ia molecules using the IA mutant mouse strain bm12 suggested that two anti-Ia monoclonal antibodies (mAbs), 25-9-17 and 34-5-3, share several features: (1) indistinguishable serologic specificity including a lack of reactivity with Iabm12, (2) binding of the same spatial epitope (cluster), and (3) definition of a cross-reactive idiotype (CRI) as defined by xenogeneic antisera. In the present study we characterize a rabbit anti-idiotype (anti-Id) to 25-9-17 by affinity chromatography, and demonstrate that it detects at least two distinct idiotopes, one shared by 25-9-17 and 34-5-3 designated CRI (25-9-17) and one unique for 25-9-17 molecules. Experiments were also undertaken to determine whether CRI (25-9-17) represents a measurable component of allogeneic humoral responses to Iab antigens. By both absorption analyses of a polyspecific antiserum and production of antigenically-restricted antisera using bm12 mice, CRI (25-9-17) was found to represent a significant proportion of the antibodies to Iab. By several criteria it was shown that the CRI (25-9-17)+ molecules were among the antibodies defining the serologic lesion of bm12 mice. In preparation for future studies to alter in vivo T-cell responses involving recognition of Ia (e.g. graft vs host disease and allogeneic transplant rejection), various immunization protocols and mouse strains were tested for induction of Id (25-9-17) following in vivo administration of various anti-idiotypic reagents. Rabbit anti-Id (25-9-17) successfully induced CRI (25-9-17) positive molecules in all strains tested regardless of IA or Ig genotype. Moreover, some of these treated mice produced antibodies to an Ia determinant missing on bm12 cells, suggesting that they recognize the same serologic determinant as mAb 25-9-17.     

Monoclonal antibodies to a CD4 peptide derivative which includes the region corresponding to an immunoglobulin CDR3: evidence of the involvement of pre-CDR3-related region in HIV-1 and host cell interaction.

A CD4 peptide of amino acid residues 68-130 [CD4(68-130)], which had the capacities to inhibit HIV-1 replication and HIV-1-induced syncytium formation, was used as an immunogen for the preparation of mAb. The mAbs prepared were classified into at least five types (I-V) in terms of their recognition sites by ELISA using various kinds of smaller CD4 peptides. Among them, the type I mAb no. 35 recognizing amino acid residues 72-84, which lies just before the region corresponding to an immunoglobulin third complementarity-determining region (CDR3), showed the strongest effects in reducing both HIV-1 infection and HIV-1-induced syncytium formation, although a large amount of no. 35 mAb was necessary to reduce such HIV-1 activities compared with those of anti-Leu-3a and OKT4A mAbs which recognize CD4 epitopes near a portion corresponding to an immunoglobulin CDR2. Western blot analysis showed that the reactivities of CD4 molecule in CD4-positive cells or sCD4 molecule with types I-V mAbs were stronger than that with anti-Leu-3a mAb. Flow cytometry showed that no. 35 mAb was faintly reactive with native CD4 molecule on cell surface at the concn showing the inhibitory effects on HIV-1 infection and syncytium formation. In addition, a smaller peptide CD4(66-92), one of the good epitope peptides for no. 35 mAb, also showed strong inhibitory effect on HIV-1 infection as well as a weaker inhibitory effect on syncytium formation. These results suggest that, in addition to the CD4 CDR2-related region, the pre-CDR3-related region is also involved in the early events of the interactions between the host cell and HIV-1.