抗鳗弧菌独特型单克隆抗体的制备及鉴定

利用具有中和活性的抗鳗弧菌单克隆抗体 4A6作为免疫原 ,通过单克隆抗体技术制备出 7株分泌抗独特型单抗的杂交瘤细胞。以ELISA竞争抑制实验及诱导Ab3的功能实验证实 ,其中 4株属于Ab2 β,有可能用于疫苗生产。     

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.     

Production and characterisation of monoclonal anti-idiotype antibody to Vibrio anguillarum

Seven monoclonal anti-idiotype antibodies (mab2) were raised against mouse monoclonal antibody (mab1) 4A6. Identification of subclass showed that 1H5, 1D1, 2B12 and 2F12 belonged to IgG2b, 2H12 and 1H12 to IgG2a and lE10 to IgG3. The titres of these mab2 ascitic fluids ranged from 1 x 10(-4)-1 x 10(-6). The capacity of the mab2 to inhibit the binding between the corresponding rabbit antiserum and Vibrio anguillarum was investigated with the competitive inhibition ELISA. The results showed that mab2 1D1, 1E10, 1H5 and 1H12 were able to inhibit this binding. Another experiment demonstrated that mab2 1D1, 1E10 and 1H5 might induce Balb/c mice to produce Ab3 and these Ab3 competed the same antigen epitopes with Ab1. These results indicate that mab2 1D1, 1E10 and 1H5 are likely to represent an internal image of V. anguillarum and may thus be described as Ab2-beta anti-idiotype antibodies. In protection experiments, Japanese flounders vaccinated with mab21D1, 1E10 and 1H5 showed significantly enhanced survival from challenge with V. anguillarum. Thus. mab21D1, 1E10 and 1H5 may have use as idiotype vaccines for fish in aquaculture.     

Protection against Streptococcus equi infection by monoclonal antibodies against an M-like protein.

We have developed an in vivo passive transfer assay using mice to identify monoclonal antibodies (mAbs) which offer protection against Streptococcus equi infection. The assay was developed using serum antibodies collected from horses convalescing from strangles. In this study, we show that a preparation of M-like protein, acid-extracted from S. equi, affords 80% protection to mice immunized with it. A number of mouse mAbs directed against a preparation of M-like protein were then assessed for their ability to passively protect mice against challenge with a lethal dose of the bacteria. Two mAbs, 1D10 and 2A6, were shown to be highly protective. It was also demonstrated, by means of a competitive enzyme immunoassay, that these mAbs recognized different epitopes in the preparation. Examination of a dose-response curve for mAbs 1D10 and 2A6 revealed that optimal levels of protection were achieved using 1 mg of either 1D10 or 2A6, or 0.5 mg 1D10 and 0.5 mg 2A6 given together. Immunological reactivity of these mAbs with a preparation of M-like protein showed that the antigens they recognized were comparable in size to some of the antigens recognized by convalescent horse serum antibodies. The role of immunoglobulin isotype in conferring protection is discussed.     

Mapping of functional epitopes of osteopontin by monoclonal antibodies raised against defined internal sequences.

Osteopontin (OPN) is a secreted protein that has been implicated in diverse physiological and pathological processes. OPN can bind to integrins, via GRGDS or SVVYGLR amino acid sequences, and to other cell surface receptors, and many of OPN's functions are likely mediated via cell adhesion and subsequent signaling. Here we developed and characterized a series of five monoclonal antibodies, raised to distinct internal peptide sequences of human OPN, and have used these sequence-specific reagents, along with the previously described anti-OPN monoclonal antibody mAb53, to map functional epitopes of OPN that are important to cell adhesion and migration. All antibodies were reactive with native as well as recombinant human OPN. One antibody (2K1) raised against the peptide VDTYDGRGDSVVYGLRS could inhibit RGD-dependent cell binding to OPN, with an efficacy comparable to that of mAb53. Furthermore, 2K1 could inhibit alpha9 integrin-dependent cell binding to OPN. The epitope recognized by 2K1 was not destroyed by thrombin digestion, whereas mAb53 has been shown to be unable to react with OPN following thrombin cleavage. The two distinct epitopes defined by 2K1 and mAb53 antibodies are closely related to the SVVYGLR cell-binding domain and the GLRSKS containing thrombin cleavage site, respectively, and are involved in cell binding and cell migration.     

Anti-idiotypic antibodies bearing the internal image of a bradykinin epitope. Production, characterization, and interaction with the kinin receptor.

mAb against bradykinin, the prototypic member of the kinin family of vasodilator peptides, were generated by somatic cell fusion. The antibodies were isotyped as IgG1, kappa-type, and their target epitopes mapped with bradykinin, lysyl-bradykinin (kallidin), kinin receptor antagonists, and fragments thereof, revealing three distinct sets of mAb, i.e., mAb against bradykinin (MBK)1, MBK2, and MBK3. Comparison of the immunologic binding affinities and the known pharmacologic binding specificities of bradykinin derivatives disclosed a striking similarity in the binding profiles of mAb MBK3 and the B2 type of the kinin receptor. Anti-idiotypic antibodies against MBK1, MBK2, and MBK3 were raised in rabbit and sheep. Inhibition and competition experiments on the level of the Ag (ligand), the idiotype, and the anti-idiotype demonstrated the mutual specificity of the network system components. Anti-idiotypic antibodies against MBK3 recognized a particular idiotope that was conformation-dependent and associated with the Ag binding site of the antibody. Binding of anti-idiotypic antibodies to the B2 receptor expressed by human foreskin fibroblasts and guinea pig ileum demonstrated that the anti-idiotypes cross-react with the corresponding receptor across species. Specific stimulation of the inositol phosphate pathway in human fibroblasts and of the PG pathway in mouse fibroblasts, respectively, and inhibition of the latter effect by the B2 kinin receptor antagonist NPC 567 indicated that the anti-idiotypes bear the internal image of a bradykinin epitope. Furthermore, antibodies of the third generation (anti-anti-idiotypic antibodies) recognized the authentic Ag, i.e., bradykinin. Hence, the anti-idiotypic approach provides powerful tools to probe for the hitherto poorly characterized B2 kinin receptor.     

Anti-idiotypic antibodies to a polyomavirus monoclonal antibody recognize cell surface components of mouse kidney cells and prevent polyomavirus infection.

Anti-idiotypic antibodies have been successfully used to identify and isolate the receptor for several cell ligands. To prepare an immunologic probe for identification of the polyomavirus receptor on mouse kidney cells, polyclonal antisera against antipolyomavirus antibodies were prepared in rabbits. Fab fragments of the previously characterized monoclonal antibody E7, which neutralizes polyomavirus infection, were used for immunization (S. J. Marriott and R. A. Consigli, J. Virol. 56:365-372, 1985). Sera containing the greatest anti-idiotype activity were identified by enzyme-linked immunosorbent assay (ELISA) and purified by a series of affinity columns. The anti-idiotypic antibodies recognized the E7 idiotope in an ELISA, and anti-idiotype binding could be inhibited by preincubation of E7 monoclonal antibody with polyomavirus virions. When mixed with anti-idiotype immunoglobulin G (IgG), E7 was no longer capable of binding or immunoprecipitating polyomavirus virions or neutralizing polyomavirus infection. Direct immunofluorescence showed anti-idiotype IgG reactivity with a cell surface component of mouse kidney cells. Anti-idiotype F(ab')2 effectively competed with polyomavirus for binding to mouse kidney cells and displayed binding kinetics similar to those of polyomavirus. Virus infection of mouse kidney cells was blocked in a dose-dependent manner following treatment of the cells with anti-idiotype IgG. The anti-idiotype identified several proteins (95, 50, and 24 to 31 kilodaltons) in an immunoblot of mouse kidney cell membrane proteins but reacted predominantly with a single 50-kilodalton protein in a radioimmunoassay. The anti-idiotype failed to react with virus proteins in three assays, including ELISA, immunoprecipitation, and immunoblotting. The implications of this work for future identification and characterization of the polyomavirus receptor on mouse kidney cells are discussed.     

A novel plant-made monoclonal antibody enhances the synergetic potency of an antibody cocktail against the SARS-CoV-2 Omicron variant

This study describes a novel, neutralizing monoclonal antibody (mAb), 11D7, discovered by mouse immunization and hybridoma generation, against the parental Wuhan-Hu-1 RBD of SARS-CoV-2. We further developed this mAb into a chimeric human IgG and recombinantly expressed it in plants to produce a mAb with human-like, highly homogenous N-linked glycans that has potential to impart greater potency and safety as a therapeutic. The epitope of 11D7 was mapped by competitive binding with well-characterized mAbs, suggesting that it is a Class 4 RBD-binding mAb that binds to the RBD outside the ACE2 binding site. Of note, 11D7 maintains recognition against the B.1.1.529 (Omicron) RBD, as well neutralizing activity. We also provide evidence that this novel mAb may be useful in providing additional synergy to established antibody cocktails, such as Evusheld™ containing the antibodies tixagevimab and cilgavimab, against the Omicron variant. Taken together, 11D7 is a unique mAb that neutralizes SARS-CoV-2 through a mechanism that is not typical among developed therapeutic mAbs and by being produced in ΔXFT Nicotiana benthamiana plants, highlights the potential of plants to be an economic and safety-friendly alternative platform for generating mAbs to address the evolving SARS-CoV-2 crisis.     

Recognition of two Dermatophagoides pteronyssinus-specific epitopes on antigen P1 by using monoclonal antibodies: binding to each epitope can be inhibited by serum from dust mite-allergic patients

Monoclonal antibodies were raised against Antigen P1, the major allergen of the house dust mite (Dermatophagoides pteronyssinus). The majority were Antigen P1 specific, isotype IgG1, and did not react with a comparable D. farinae allergen. These antibodies bound 38 to 50% of 125I Antigen P1 in antigen-binding assays (titer greater than or equal to 1/1,000,000), and the quantities of IgG antibody in ascites were 2 to 4 logs greater than those in polyclonal mouse antiserum or in serum from a mite-allergic patient. Two IgM antibodies showed weak binding to Antigen P1 but reacted strongly with D. pteronyssinus in enzyme immunoassay (titer greater than or equal to 1/100,000). Assessments of the specificity of the IgG antibodies by using two inhibition radioimmunoassays suggested that they were directed against two different epitopes. Antibodies 10B9 F6 and 5H8 C12 were purified by preparative isoelectric focusing (isoelectric points of pI 6.25 and 7.4, respectively) and radiolabeled with 125I. Cross-inhibition experiments, using ascites dilutions to inhibit binding of each radiolabeled antibody to Antigen P1, confirmed that these antibodies recognized two distinct epitopes. Analysis of antibodies from 39 clones/hybrids showed that the majority were directed against the same epitopes as either 10B9 F6 or 5H8 C12 (3 out of 39 [8%] and 29 out of 39 [74%], respectively). None of the monoclonal antibodies significantly inhibited (greater than 10%) human IgE binding to Antigen P1 in the radioallergosorbent test. However, 12 of 14 sera from mite allergic patients inhibited binding by the monoclonal antibodies. One serum from a mite-allergic patient inhibited binding of both 10B9 F6 and 5H8 C12 by greater than 85% and showed parallel inhibition curves. The results suggest that these monoclonal antibodies could be used to assay Antigen P1 in both D. pteronyssinus and house dust extracts. It should also be possible to use monoclonal antibodies in inhibition assays to define the antigenic/allergenic determinants recognized by human IgG and IgE antibodies on this mite allergen.     

Immunization with an anti-idiotypic antibody against the broadly lipopolysaccharide-reactive antibody WN1 222-5 induces Escherichia coli R3-core-type specific antibodies in rabbits

The mouse monoclonal antibody (mAb) WN1 222-5 recognizes a carbohydrate epitope in the inner core region of LPS that is shared by all strains of Escherichia coli and Salmonella enterica and is able to neutralize their endotoxic activity in vitro and in vivo. Immunization of mice with mAb WN1 222-5 yielded several anti-idiotypic mAbs one of which (mAb S81-19) competitively inhibited binding of mAb WN1 222-5 to E. coli and Salmonella LPS. After immunization of rabbits with mAb S81-19, the serological responses towards LPS were characterized at intervals over two years. Whereas the serological response against the anti-idiotype developed as expected, the anti-anti-idiotypic response against LPS developed slowly and antibodies appeared after 200?d that bound to E. coli LPS of the R3 core-type and neutralized its TNF-α inducing capacity for human peripheral mononuclear cells. We describe the generation of a novel anti-idiotypic antibody that can induce LPS core-reactive antibodies upon immunization in rabbits and show that it is possible, in principle, to obtain LPS neutralizing antibodies by anti-idiotypic immunization against the mAb WN1 222-5. The mimicked epitope likely shares common determinants with the WN1 222-5 epitope, yet differences with respect to either affinity or specificity do exist, as binding to smaller oligosaccharides of the inner core was not observed.     

The antigen identified by a mouse monoclonal antibody raised against human renal cancer cells is the adenosine deaminase binding protein

The antigen recognized by a mouse monoclonal antibody (mAb S27) raised against a human renal cancer cell line has been identified as the adenosine deaminase binding protein. mAb S27 immunoprecipitates binding protein purified from a soluble fraction of human kidney. It also recognizes the mature 120,000-dalton membrane form of binding protein from [35S]methionine-labeled human fibroblasts, HepG2 cells, and the renal cancer cell line against which the antibody was raised. A rabbit polyclonal antibody raised against purified kidney binding protein completely precipitates mAb S27-reactive material from labeled membrane extracts. mAb S27 does not precipitate the initially synthesized 110,000 molecular weight precursor of binding protein in fibroblasts and recognizes only a small portion of binding protein precursor in labeled HepG2 cells suggesting that the antigenic determinant recognized by mAb S27 may be a post-translational modification present on the mature form of binding protein or that mAb S27 recognizes molecules in a certain conformation. Glycopeptides derived from purified soluble kidney binding protein or exogenously added adenosine deaminase do not inhibit the immunoprecipitation of binding protein by mAb S27, indicating that the mature oligosaccharide chains of binding protein are not the determinant recognized by mAb S27 and that bound adenosine deaminase does not mask the antigenic sites on binding protein. The fact that monoclonal antibody S27, previously shown (Ueda, R., Ogata, S., Morissey, D. M., Finstad, C. L., Szkudlavek, J., Whitmore, W. F., Oettgen, H. F., Lloyd, K. O., and Old, L. J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5122-5126) to detect a cell surface antigen on cultured renal cancer cells, is directed against the adenosine deaminase binding protein confirms and extends the earlier observation (Andy, R.J., and Kornfeld, R. (1982) J. Biol. Chem. 257, 7922-7925) that binding protein is located on the cell surface.     

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     

Rheumatoid factors may bear the internal image of the Fc gamma-binding protein of herpes simplex virus type 1

Affinity-purified rheumatoid factors (RF) from 20 patients with rheumatoid arthritis were tested for their reactivity with the mAb II-481 against glycoprotein E (gE), the Fc gamma-binding protein of HSV-1, as well as with a panel of mAb against human Fc gamma R. All RF bound to mAb II-481 in preference to mAb IV.3 (anti-human Fc gamma RII) or MOPC 141 (control mAb) which belong to the same IgG2b subclass. Five RF showed strong reactivity with II-481. No significant reactivity was observed between RF and mAb against human Fc gamma R. Non-RF human IgM did not react with any of the mAb. Clear-cut binding to II-481 was also seen with monoclonal IgM-RF derived from MRL/1 mice (mRF-2). The reaction between RF and II-481 was completely inhibited by human IgG. It was also inhibited by BHK cell extract infected with HSV-1, and with purified gE. II-481 inhibited the binding of human IgG Fc to the infected cell extract, confirming that II-481 recognizes the Fc-binding site on gE. II-481 did not react directly with human IgG or Fc of IgG. mAb to human IgG2 showed stronger binding to II-481 than to MOPC 141, suggesting II-481 has conformational similarity to human IgG H chain. These results suggest that at least some RF bear the "internal image" of HSV-1 Fc gamma-binding protein and support the hypothesis that some RF may be generated as anti-idiotype antibodies against antiviral antibodies.     

Induction of an immune response through the idiotypic network with monoclonal anti-idiotype antibodies in the carcinoembryonic antigen system.

Anti-idiotype antibodies can mimic the conformational epitopes of the original antigen and act as antigen substitutes for vaccination and/or serological purposes. To investigate this possibility concerning the tumor marker carcinoembryonic antigen (CEA), BALB/c mice were immunized with the previously described anti-CEA monoclonal antibody (MAb) 5.D11 (AB1). After cell fusion, 15 stable cloned cell lines secreting anti-Ids (AB2) were obtained. Selected MAbs gave various degrees of inhibition (up to 100%) of the binding of 125I-labeled CEA to MAb 5.D11. Absence of reactivity of anti-Id MAbs with normal mouse IgG was first demonstrated by the fact that anti-Id MAbs were not absorbed by passage through a mouse IgG column, and second because they bound specifically to non-reduced MAb 5.D11 on Western blots. Anti-5.D11 MAbs did not inhibit binding to CEA of MAb 10.B9, another anti-CEA antibody obtained in the same fusion as 5.D11, or that of several anti-CEA MAbs reported in an international workshop, with the exception of two other anti-CEA MAbs, both directed against the GOLD IV epitope. When applied to an Id-anti-Id competitive radioimmunoassay, a sensitivity of 2 ng/ml of CEA was obtained, which is sufficient for monitoring circulating CEA in carcinoma patients. To verify that the anti-Id MAbs have the potential to be used as CEA vaccines, syngeneic BALB/c mice were immunized with these MAbs (AB2). Sera from immunized mice were demonstrated to contain AB3 antibodies recognizing the original antigen, CEA, both in enzyme immunoassay and by immunoperoxidase staining of human colon carcinoma. These results open the perspective of vaccination against colorectal carcinoma through the use of anti-idiotype antibodies as antigen substitutes.     

Identification of a Conserved B-Cell Epitope on Duck Hepatitis A Type 1 Virus VP1 Protein

BackgroundThe VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized.

Methods and Results

To characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope ¹⁷³LPAPTS¹⁷⁸ is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1–positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope.

Conclusions and Significance

We identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1.     

Characterization of binding of four monoclonal antibodies to the human ovarian adenocarcinoma cell line HEY

Four mouse monoclonal antibodies (mAb) (10B, IgG1; 8C, IgG2a; M2A, IgG2a; M2D, IgG2b) were characterized with respect to their binding to the ovarian adenocarcinoma cell line HEY, using displacement assays and Scatchard plot analyses. The four mAb reacted with different antigens on the surface of HEY cells, with affinity constants ranging from 1 X 10(9) to 3 X 10(9) M-1. The number of binding sites per cell for each antibody was approximately 2 X 10(4). mAb 8C and M2D remained associated with the cell surface following binding to their respective antigens, while mAb 10B was rapidly internalized, with 50% of the bound mAb being lost from the cell surface during 4 h of incubation at 37 degrees C. These different binding characteristics of the mAb may influence their ability to target radioactivity and cytotoxic drugs to HEY cells.     

A monoclonal anti-idiotypic antibody against a human monoclonal IgM with specificity for myelin-associated glycoprotein

A human monoclonal IgM lambda antibody, directed against MAG, obtained from a patient with polyneuropathy associated with a gammopathy, was used as an immunogen to generate mouse monoclonal anti-idiotype antibodies. One hybridoma antibody, designated A8F2, reacts uniquely with the M-IgM of the patient, shows high affinity binding to the patient's M-IgM, and dose-dependently inhibits binding of the patient's M-IgM to its specific antigen MAG. Thus, A8F2 is a monoclonal anti-idiotype antibody that recognizes a region of the MAG binding site of the patient's IgM. Use of this anti-idiotype antibody in a competition RIA revealed the presence of naturally occurring anti-idiotype in the patient's serum. Because anti-idiotype antibodies may be part of a mechanism for down-regulation of antibody production, the use of A8F2 to induce a specific immunosuppression should be considered.     

Overcoming Antigenic Diversity by Enhancing the Immunogenicity of Conserved Epitopes on the Malaria Vaccine Candidate Apical Membrane Antigen-1

Malaria vaccine candidate Apical Membrane Antigen-1 (AMA1) induces protection, but only against parasite strains that are closely related to the vaccine. Overcoming the AMA1 diversity problem will require an understanding of the structural basis of cross-strain invasion inhibition. A vaccine containing four diverse allelic proteins 3D7, FVO, HB3 and W2mef (AMA1 Quadvax or QV) elicited polyclonal rabbit antibodies that similarly inhibited the invasion of four vaccine and 22 non-vaccine strains of P. falciparum. Comparing polyclonal anti-QV with antibodies against a strain-specific, monovalent, 3D7 AMA1 vaccine revealed that QV induced higher levels of broadly inhibitory antibodies which were associated with increased conserved face and domain-3 responses and reduced domain-2 response. Inhibitory monoclonal antibodies (mAb) raised against the QV reacted with a novel cross-reactive epitope at the rim of the hydrophobic trough on domain-1; this epitope mapped to the conserved face of AMA1 and it encompassed the 1e-loop. MAbs binding to the 1e-loop region (1B10, 4E8 and 4E11) were ∼10-fold more potent than previously characterized AMA1-inhibitory mAbs and a mode of action of these 1e-loop mAbs was the inhibition of AMA1 binding to its ligand RON2. Unlike the epitope of a previously characterized 3D7-specific mAb, 1F9, the 1e-loop inhibitory epitope was partially conserved across strains. Another novel mAb, 1E10, which bound to domain-3, was broadly inhibitory and it blocked the proteolytic processing of AMA1. By itself mAb 1E10 was weakly inhibitory but it synergized with a previously characterized, strain-transcending mAb, 4G2, which binds close to the hydrophobic trough on the conserved face and inhibits RON2 binding to AMA1. Novel inhibition susceptible regions and epitopes, identified here, can form the basis for improving the antigenic breadth and inhibitory response of AMA1 vaccines. Vaccination with a few diverse antigenic proteins could provide universal coverage by redirecting the immune response towards conserved epitopes.     

Shared idiotypes on anti-DNA and anti-poly (ADP-ribose) antibodies

Hed 10 is a ssDNA-specific mAb derived from an NZB/W autoimmune mouse. ADP 1 is a poly(ADP-ribose)-specific mAb derived from a C57BL/6 mouse. Rabbit anti-idiotypic sera were raised against Hed 10 and ADP 1. By affinity chromatography it was shown that at least 20 to 30% of DNA-binding antibodies contained these idiotypes. The sera were also used to evaluate idiotypic cross-reactivity among 26 diverse, predominantly anti-nucleic acid, murine mAb. Each serum bound directly to several mAb and in addition inhibited the binding of several antibodies to their appropriate Ag. The anti-Hed 10 serum detected an idiotype which was restricted to antibodies that bound to poly(dT) and/or poly(ADP-ribose). The anti-ADP 1 serum detected a more widely distributed idiotype contained in antibodies which bound to a variety of nucleic acids. Although both sera bound directly to Hed 10, only the anti-Hed 10 serum could compete for the binding of Hed 10 to poly(dT). On the other hand, both sera could compete for the binding of ADP 1 to poly(ADP-ribose) as well as bind directly to ADP 1. In addition anti-ADP 1 serum appeared to enhance, rather than inhibit, the binding of one mAb to native calf thymus DNA and poly[d(AT)] but had no effect on the binding to several ssDNA. These results demonstrate that many antibodies which recognize DNA and poly(ADP-ribose) have shared idiotypes. This may be of relevance to the development of autoimmunity because poly(ADP-ribose) is ubiquitous and immunogenic.     

Isolation and function of a human endothelial cell C1q receptor

It has been shown previously that cultured human venous and arterial endothelial cells (EC) bind C1q in a time- and dose-dependent manner. Cultured human endothelial cells express an average number of 5.2 x 10(5) binding sites/cell. In the present study the putative receptor for C1q (C1qR) was isolated from the membranes of 1-5 x 10(9) human umbilical cord EC by affinity chromatography on C1q-Sepharose. During isolation, C1qR was detected by its capacity to inhibit the lysis of EAC1q in C1q-deficient serum. The eluate from C1q-Sepharose was concentrated, dialysed and subjected to QAE-A50 chromatography and subsequently to gel filtration on HPLC-TSK 3000. C1qR filtered at an apparent molecular weight of 60 kDa. Purified C1qR exhibited an apparent molecular weight of 55-62 kDa in the unreduced state and a molecular weight of 64-68 kDa in reduced form. Two IgM monoclonal antibodies (mAb) D3 and D5 were raised following immunization of mice with purified receptor preparations. Both monoclonal antibodies increased the binding of (125)I-C1q to endothelial cells but F(ab')(2) anti-C1qR mAb inhibited the binding of a(125)I-C1q to EC in a dosedependent manner. The D3 mAb recognized a band of 54-60 kDa in Western blots of membranes of human EC and polymorphonuclear leukocytes. Previously, the authors showed that C1q induces the binding of IgM-containing immune complexes to EC. Therefore, it was hypothesized that during a primary immune response generation of IgM-IC may occur, resulting in binding and activation of C1, dissociation of activated C1 by C1 inhibitor and subsequent interaction of IgM-IC bearing C1q with EC-C1qR.