Neutralization of tetanus toxin by distinct monoclonal antibodies binding to multiple epitopes on the toxin molecule.

Fifty-seven hybridomas producing antibodies to tetanus toxoid or to the Ibc or B-IIb fragment of the toxin were isolated independently. Competitive inhibition studies demonstrated that monoclonal antibodies from mice immunized with the toxoid bound to at least 20 different epitopes on the toxoid molecule. Similar competitive binding studies revealed eight distinct epitopes on the B-IIb fragment and three to five epitopes on the Ibc fragment of the toxin. Neutralization of toxicity was effected by nine distinct monoclonal antibodies from hybridomas of toxoid-immunized mice and by one monoclonal antibody from B-IIb-immunized mice. Mixtures of two, three, and four different monoclonal antibodies in a variety of combinations exerted a synergistic effect of ca. 200-fold over that observed with individual monoclonal antibodies, indicating that efficient neutralization may involve the simultaneous binding of at least two antibody molecules to different specific regions of the toxin molecule. Only one toxoid-induced monoclonal antibody failed to bind to tetanus toxin. All neutralizing antibodies bound to epitopes on the heavy chain of tetanus toxin. Six of these were directed toward epitopes on the NH2-terminal half, whereas four bound to epitopes on the carboxy-terminal half of the heavy chain. Only one monoclonal antibody bound preferentially to the light chain, but two other monoclonal antibodies appeared to bind to both chains, indicating some homology between these two chains.     

Autologous Mixed Lymphocyte Responses in Experimentally-induced Arthritis of the Lewis Rat

Lewis rats develop immune-mediated arthritis following injection with a variety of agents including bovine type II collagen (bCII), mycobacteria, muramyl dipeptide and CP20961. Since susceptibility to experimentally-induced arthritis has been linked to the genes encoding the major histocompatibility complex, it is hypothesized that antigen presentation to autoreactive T-cells is a critical event in the pathogenesis of disease. T-cells, isolated from Lewis rats immunized with bCII or mycobacteria, were co-cultured with splenic or thymic antigen presenting cells (APC) and proliferative responses to antigen were assessed by 3 H-thymidine incorporation. T-cell proliferation was observed upon culture with APC without requiring the addition of antigen. T-cells from rats injected with non-immunogenic adjuvants also demonstrated an increased autologous MLR compared to T-cells from non-injected animals. In contrast, T-cells from animals immunized with non-arthritogenic antigens, including ovalbumin or tetanus toxoid, proliferated only when co-cultured with specific antigen-pulsed APC. These results suggest that immunization with arthritogens activates a population of self-reactive T-cells, which respond in an autologous MLR. We propose that these autoreactive T-cells recognize endogenously-derived self peptides rather than peptides derived from a joint autoantigen.     

Autologous mixed lymphocyte responses in experimentally-induced arthritis of the Lewis rat

Lewis rats develop immune-mediated arthritis following injection with a variety of agents including bovine type II collagen (bCII), mycobacteria, muramyl dipeptide and CP20961. Since susceptibility to experimentally-induced arthritis has been linked to the genes encoding the major histocompatibility complex, it is hypothesized that antigen presentation to autoreactive T-cells is a critical event in the pathogenesis of disease. T-cells, isolated from Lewis rats immunized with bCII or mycobacteria, were co-cultured with splenic or thymic antigen presenting cells (APC) and proliferative responses to antigen were assessed by 3H-thymidine incorporation. T-cell proliferation was observed upon culture with APC without requiring the addition of antigen. T-cells from rats injected with non-immunogenic adjuvants also demonstrated an increased autologous MLR compared to T-cells from non-injected animals. In contrast, T-cells from animals immunized with non-arthritogenic antigens, including ovalbumin or tetanus toxoid, proliferated only when co-cultured with specific antigen-pulsed APC. These results suggest that immunization with arthritogens activates a population of self-reactive T-cells, which respond in an autologous MLR. We propose that these autoreactive T-cells recognize endogenously-derived self peptides rather than peptides derived from a joint autoantigen.     

Induction of primary antigen-specific immune reponses in SCID-hu-PBL by coupled T-B epitopes.

Adoptive transfer of human lymphoid cells into immunodeficient (SCID) mice lacking the ability to functionally rearrange T- and B-cell receptor genes constitutes a unique model to study and manipulate human immunocytes. We have investigated this model for the purpose of generating an antigen-specific primary humoral immune response. Peripheral blood lymphocytes (PBL) derived from blood donors were used to repopulate SCID mice, which subsequently were immunized with different B-cell epitopes coupled to either tetanus toxoid (TT), or to a promiscuous helper epitope of TT, or by incorporating the antigens into a liposome construct. By recruiting the necessary T-cell help found in the T-cell memory compartment against TT, primary immune responses were obtained against the hapten dinitrophenyl (DNP), the V3 loop peptide derived from glycoprotein (gp120) (HIV-1), the melanoma-associated GD2 ganglioside and ovine submaxillary mucin. The primary immune response against the GD2 ganglioside was induced by incapsulating TT into GD2-containing liposomes. These liposome constructs also allowed us to induce a high human IgG serotitre (3000-4000) against this normally not very immunogenic ganglioside.     

Immunogenicity and protection in small-animal models with controlled-release tetanus toxoid microparticles as a single-dose vaccine.

Tetanus toxoid (TT) was encapsulated in microparticles prepared from polylactide-co-glycolide polymers by a solvent-evaporation technique. Combinations of small- and large-sized microparticles with controlled-release characteristics were used to immunize Sprague-Dawley rats, and the antibody responses were monitored for 1 year. For comparison, control groups of rats were immunized at 0, 1, and 2 months with TT adsorbed to alum. The antibody responses generated by the TT entrapped in microparticles were comparable to those generated by TT adsorbed to alum in control groups from 32 weeks onwards. Microparticles with a single entrapped antigen (TT) induced better antibody responses than microparticles with two antigens (TT and diphtheria toxoid) entrapped simultaneously. A combination vaccine consisting of TT adsorbed to alum and also entrapped in microparticles gave the best antibody responses. In an inhibition assay designed to determine the relative levels of binding of antisera to the antigens, the sera from the microparticle- and the alum-immunized animals showed comparable levels of binding. In addition, in a passive-challenge study with mice, TT adsorbed to alum and TT entrapped in microparticles provided equal levels of protection against a lethal challenge with tetanus toxin. An intradermal-challenge study was also performed with rabbits, which showed similar levels of protection in sera from alum- and microparticle-immunized animals at 4, 12, and 32 weeks after immunization.     

A群脑膜炎球菌多糖结合物的免疫原性研究

目的：制备A群脑膜炎球菌多糖（PS）结合物，观察其免疫原性。方法：A群脑膜炎球菌多糖经溴化氰活化后共价接上己二酰肼手臂，在碳二亚胺催化下与精制破伤风类毒素（TT）偶联剂备结合物。单独多糖、多糖结合物和TT按相同程序免疫NIH小鼠，用ELISA法检测小鼠血清抗多糖抗体滴度和特异性及抗TT抗体滴度。结果：用上述实验方法制备的结合物具有多糖和蛋白质的抗原性，免疫小鼠可诱导高滴度的血清抗多糖IgG抗体，免疫2针后高滴度抗体至少持续存在3周。血清抗体可中和多糖。加强免疫提示有免疫记忆性。结合物还可诱导产生一定水平的TT抗体。结论：A群脑膜炎球菌多糖结合到TT上可明显增强多糖在小鼠中的免疫原性，为制备结合疫苗并研究其对婴幼儿的免疫原性提供了实验基础。     

Polyclonal cell activity of a repeat peptide derived from the sequence of an 85-kilodalton surface protein of Trypanosoma cruzi trypomastigotes.

Some in vitro and in vivo biological activities of an octadecapeptide derived from an 85-kDa surface protein of Trypanosoma cruzi trypomastigote were studied. The peptide coupled to a carrier protein induced the proliferative response of lymph node cells from mice immunized with various antigens. Moreover, sera from mice immunized with the coupled peptide were found to contain antibodies against a number of self and nonself antigens: fibronectin, bovine serum albumin, myosin, tetanus toxoid, ovalbumin, keyhole limpet hemocyanin, and DNA. These results are discussed in the context of Chagas' disease immunopathology.     

14型肺炎球菌荚膜多糖-破伤风类毒素结合疫苗研究

目的：探讨制备肺炎球菌荚膜多糖（PNCPS）－蛋白结合疫苗适宜条件。方法：采用碳二亚胺法将14型PNCPS与破伤风类毒素（TT）结合，将结合物免疫NIH小鼠，用ELISA法检测小鼠血清中抗14例PNCPS的IgG抗体滴度。结果：结合反应产率和结合物中多糖，蛋白含量的测定显示试验成功合成了14型PNCPS－TT结合物，该结合物具有14型PNCPS的血清学特异性，将之免疫小鼠诱生的抗14型PNCPS特异性IgG抗水平显著高于单纯14型PNCPS。结论：试验成功地合成了14型PNCPS－TT结合物，本试验采用的结合方法可行。     

Production of antineutrophil cytoplasm antibodies derived from circulating B cells in patients with systemic vasculitis

The pathogenesis of systemic vasculitis is complex and is likely to involve many mechanisms. In certain systemic vasculitides, autoimmunity plays an important role with autoantibodies developing towards neutrophils, which are termed antineutrophil cytoplasm antibodies (ANCA). There is a growing body of evidence that T cells may contribute to the pathogenesis of ANCA-associated vasculitides. A system was set up to determine whether B cells require T cell help to produce antibodies in a peripheral blood lymphocyte (PBL) culture system enriched for B cells and dendritic cells (DC). As a control, tetanus toxoid (TT) antibody production was detected from individuals not recently immunized with tetanus vaccine when stimulated with TT antigen. Proteinase 3 (PR3) and myeloperoxidase (MPO) antibodies were produced from B cell and DC enriched cultures prior to the addition of antigen in some ANCA-positive patients with high ANCA titres, but not from patients with low ANCA titres or controls. PBMC from individuals recently immunized with tetanus vaccine were also maximally stimulated in that addition of antibody did not enhance antibody production. We conclude that this system supports a role for T cell help in the production of TT antibodies in individuals not immunized recently with tetanus vaccine. However, in patients with ANCA-associated vasculitis and controls recently immunized with tetanus vaccine, circulating B cells are apparently spontaneously producing autoantibody, possibly reflecting a system already maximally driven in vivo, and therefore masking underlying potential T cell-B cell collaboration. Such B cells may be less responsive to regulatory stimuli in vivo.     

Production and characterization of monoclonal antibodies to tetanus toxin

Monoclonal antibodies against tetanus toxin were produced to obtain highly specific antisera. Ten hybridoma cell lines producing monoclonal antibodies were derived from the fusion of rat myeloma cells and spleen cells from rats immunized with tetanus toxoid. Eight produced monoclonal antibodies specific for determinants on toxin and toxoid, whereas two were specific only for determinants on the toxoid. The antibodies produced by hybridomas were characterized by determination of the class of light and heavy chain components, epitope specificity, toxin neutralization, and subunit specificity. All of the antibodies contained kappa light chain, eight contained the gamma 1 heavy chain, and the remaining two contained the gamma 2a heavy chain. Five distinct epitopes were indicated by competition assay of paired monoclonal antibodies, and 4 of the 10 monoclonal antibodies neutralized the in vivo activity of tetanus toxin. The four neutralizing monoclonal antibodies and one other were specific for the C fragment of the heavy chain of the toxin molecule.     

Chimaeric monoclonal antibodies encoded by the human VH26 gene from naïve transgenic mice display a wide range of antigen-binding specificities.

To elucidate the molecular basis for the ability of antibodies encoded by the human VH26 heavy-chain variable region gene to react with diverse antigens, we have generated 34 hybridomas secreting chimaeric monoclonal antibodies (human mu heavy chain/mouse light chains) from transgenic mice. The transgenic mice carry an immunoglobulin minilocus containing the human VH26 gene, human DH and JH gene segments, and genes encoding the human C mu region. The minilocus in these animals undergoes functional rearrangement resulting in the production of chimaeric antibodies in which human mu heavy chains utilizing the VH26 gene are paired with mouse kappa or lambda light chains. The hybridomas described in this study were generated from naïve animals and were selected solely on the basis of human mu-chain expression. The antibodies described have covalently attached mouse light chains and are multimeric in structure. The binding properties of the antibodies were examined using a panel of both self- and foreign antigens using enzyme-linked immunosorbent assays, agglutination or radio-immunoprecipitation assays and immunofluorescence. Chimaeric immunoglobulins from 21 of the 34 hybridoma clones (61.7%) reacted with one or more antigens, of which 13 (38.2%) reacted with more than two antigens. These studies demonstrate that the VH26 gene, in combination with human DH and JH gene segments, and mouse light-chain genes, is able to encode antibodies with a wide range of ligand-binding specificities. These findings have important implications in the context of the possible origins of autoantibodies encoded by VH26 which may play a role in the pathogenesis of a number of autoimmune conditions.     

Systemic immune response after mucosal immunization in patients with IgA nephropathy

Increased IgA production has been proposed as a portion of the etiology of IgA nephropathy. Indirect human data suggest that IgG and complement may be equally important. We have immunized 17 patients with IgA nephropathy and 27 controls with tetanus toxoid. They were nasally immunized and, 2 weeks later, received an im booster immunization. This protocol has been shown to result in an increased serum IgA1 antibody response to tetanus toxin (TT). Patients had higher serum IgG antibodies to TT before and after the im immunization than did controls (pre, 42 vs 13 U; post, 155 vs 71 U;P=0.004). Patients also had a greater increase in serum IgG antibodies (118 vs 58;P=0.02). After the im TT, patients had lower levels of serum IgA1 antibody to TT (115 vs 180;P=0.005) but the change in IgA1 antibodies was not significant. These data suggest that patients with IgA nephropathy may produce inappropriately large amounts of serum IgG antibodies to antigens encountered in the upper respiratory tree. Such antigens also induce a serum IgA1 response. Such a response could result in the formation of potentially nephritogenic immune complexes containing IgG, IgA1, and C3.     

Polyspecific human and murine antibodies to diphtheria and tetanus toxoids and phospholipids.

Human-human hybridomas produced from lymphocytes of normal individuals yielded seven clones producing monoclonal antibody reacting with tetanus toxoid. Three of these antibodies cross-reacted with diphtheria toxoid. These three and two others also reacted with cardiolipin and two with other phospholipids. One of the seven antibodies reacted with tetanus and diphtheria toxoids, cardiolipin and single-stranded DNA. All seven antibodies were IgM. To examine further this unusual cross-reactivity serum antibodies from patients with SLE and healthy individuals were affinity-purified to yield diphtheria toxoid antibodies. Six out of nine of these anti-diphtheria preparations contained IgG antibodies which cross-reacted with tetanus toxoid and two of these also reacted with cardiolipin; four preparations cross-reacted with DNA. Anti-cardiolipin and anti-DNA cross-reactivity were found in preparations from both normal and SLE sera. Similar cross-reactivities were demonstrated using four mouse monoclonal IgM antibodies raised against phospholipids. All four of these antibodies reacted with both cardiolipin and tetanus toxoid and two also reacted with diphtheria toxoid and DNA. Using a thiocyanate elution procedure, it was shown that the cross-reactivity of the monoclonal antibodies was not related to their relative affinities. The results clearly indicate that cross-reactive epitopes occur on routinely used toxoid vaccines and self antigens. Antibodies which bind to these cross-reactive epitopes are common and are not restricted in isotype, affinity or species of origin.     

Optimizing oral vaccines: induction of systemic and mucosal B-cell and antibody responses to tetanus toxoid by use of cholera toxin as an adjuvant.

Cholera toxin (CT) is an effective mucosal antigen and acts as an adjuvant when given orally with various antigens; however, few studies have compared the levels of antibody responses to CT and coadministered protein in systemic and mucosal tissues. In this study, we used tetanus toxoid (TT) for assessment of immune responses. Time course and dose-response studies established that 250 micrograms of TT given orally with 10 micrograms of CT three times at weekly intervals induced high serum and gastrointestinal tract anti-TT and anti-CT antibody responses. Oral immunization with TT alone induced no detectable mucosal immunoglobulin A (IgA) antibodies in fecal extracts and only weak serum IgG anti-TT responses. The coadministration of CT and TT induced peak serum IgG anti-TT responses following two oral doses that remained constant after the third oral immunization, while optimal mucosal IgA responses were seen after the third oral immunization. The serum anti-TT response obtained with CT and TT proved protective against TT challenge (100 minimum lethal doses), whereas mice orally given CT or TT alone died. Antigen-specific B-cell responses were assessed with an isotype-specific Elispot assay of isolated lymphoid cells from the spleen, Peyer's patches, and the small intestinal lamina propria. Interestingly, approximately fourfold-higher numbers of IgA anti-CT than of anti-TT antibody-producing (spot-forming) cells occurred in lymphocytes from the lamina propria of mice orally immunized with both TT and CT. The adjuvant CT did not induce polyclonal B-cell responses in mice given CT by the oral route, since no significant differences in total numbers of B cells producing IgA, IgG, or IgM were found compared with the numbers in mice given TT alone. The results clearly indicate that serum and mucosal antibody responses develop with different kinetics and that protective TT-specific antibody responses are generated in the systemic compartment when TT is administered with CT via the oral route.     

Effect of thymosin alpha one on specific antibody response and susceptibility to infection in young and aged mice

The antibody response to a variety of antigens has been shown to diminish with age. We investigated the capacity for Thymosin Alpha One (T alpha 1) treatment to augment antibody production in tetanus toxoid (TT) and pneumococcal capsular polysaccharide (PN) inoculated young and old mice. We also measured survival of these immunized mice after aerosol exposure to Streptococcus pneumoniae. As predicted antibody response to TT, but not PN, was significantly reduced in the old animals and T alpha 1 augmented antitetanus antibody in both young and old mice. T alpha 1 did not have an effect on anti pneumococcal antibody production. All mice that had received PN did have an antibody response, yet survival after exposure to the organism was strikingly less in the old animals. Our data support the contention that antibody response to T-dependent antigens (such as tetanus toxoid) falls with aging but can be reconstituted somewhat by thymic factors. Furthermore, for T-independent antigen (such as pneumococcal capsular antigens) the age-related changes are less evident. In the latter situation, the presence of a brisk antibody response after vaccination was not sufficient to prevent pneumonia and death in old animals.     

抗巨细胞病毒早晚期抗原单克隆抗体制备及在病毒培养物鉴定中的初步应用

目的 制备抗CMV早晚期抗原单克隆抗体并在病毒培养物鉴定中初步应用.方法 CMV感染MRC-5细胞24h、48h和72h后甲醛灭活的可溶性抗原免疫BALB/c小鼠,小鼠脾细胞与骨髓瘤细胞NS-1进行融合.酶免法筛查阳性杂交瘤并有限性稀释法进行克隆.免疫荧光及印迹对筛选后的克隆进行鉴定,最终获得的杂交瘤制备腹水并使用Protein G进行纯化.纯化后单抗应用于CAP室间质评标本以及临床标本CMV培养物的鉴定.结果 3只小鼠脾细胞与骨髓瘤细胞融合后初步筛查出约110株阳性克隆.剔除与其它抗原有交叉反应、抗体效价低、非全覆盖早晚期抗原的克隆最终获得抗CMV单抗杂交瘤1株,即23B5-1.免疫荧光显示23B5-1株单抗与CMV感染后3h-120h的MRC-5细胞均反应,即覆盖即刻、早期和晚期抗原.免疫印迹试验显示23B5-1株单抗与CMV抗原25KD-50KD之间的5个蛋白条带结合.23B5-1株单抗免疫球蛋白亚型为IgG1.Protein G纯化腹水后的单抗效价≥1∶12800.纯化单抗染色鉴定6份室间质评及10份临床标本CMV培养物全部符合.结论 初步应用显示制备的抗CMV早晚期抗原单克隆抗体性能良好.     

Evidence for CD4+ T cell responses common to Plasmodium falciparum and recall antigens

In individual donors which have never been exposed to malaria parasites, the CD4+ T cell precursor frequencies for tetanus toxoid (TT) and Plasmodium falciparum responses are similar (range 1:850- 1:4800). Limiting dilution cultures set up in response to P. falciparum trophozoites can be re-stimulated with the same stage of the parasite or TT and respond with similar frequencies. A substantial overlap in the responses to different agents was confirmed in suicide selection experiments where cells responding to malaria parasite, TT or influenza virus antigens were deleted using the cell cycle inhibitor cytosine arabinoside (Ara-C). The responses of the remaining cells to P. falciparum were almost completely abrogated and only weak responses were observed to different recall antigens (0.2-21% of untreated control). Little or no effect was observed on the responses to superantigen or mitogen. Furthermore, in contrast to superantigen, the observed responses to TT and Plasmodium were polyclonal, the blastoid cells generated reacting with a range of anti-TCR Vbeta antibodies with little preferential usage.      

Rearrangement of the human heavy chain variable region gene V3-23 in transgenic mice generates antibodies reactive with a range of antigens on the basis of VHCDR3 and residues intrinsic to the heavy chain variable region

To formulate a 'logic' for how a single immunoglobulin variable region gene generates antibodies with different antigen specificity and polyreactivity, we analysed chimeric antibodies produced in transgenic mice carrying the germ-line human V3-23 gene, multiple diversity (D) and joining (J) gene segments. Hybridomas producing antibodies encoded by the V3-23 gene in combination with different mouse Vkappa genes were obtained by fusion of splenocytes from transgenic mice. All antibodies had human mu-chains and mouse light chains, were multimeric in structure and expressed the human V3-23 gene. Nucleotide sequence analyses of genes encoding the heavy and light chains of 12 antibodies in relation to antigen specificity highlighted the importance of heavy chain variable region CDR3 in determining reactivity with different antigens. However, the results also suggest that non-CDR3 sequences intrinsic to the V3-23 gene itself may be involved in, or determine, the binding of the chimeric antibodies to some of the antigens tested in the current study.     

Measurement of human and mouse anti-tetanus antibodies and isotype analysis by ELISA

A rapid and sensitive enzyme immunoassay (ELISA) was developed for the quantitation of anti-tetanus antibodies. This technique was used to measure antibody levels in the plasma of immunized donors, in human anti-tetanus IgG preparations and in human and mouse hybridomas producing monoclonal antibodies to tetanus toxoid. The assay was capable of detecting antibody levels as low as 5 X 10(-4) IU/ml. By inclusion of an extra step involving antibodies to mouse Ig isotypes, a sandwich enzyme immunoassay (SEI) was developed which permitted determination of the Ig isotype of mouse anti-tetanus antibodies including tetanus-specific mouse monoclonal antibodies. SEI confirmed Protein A-Sepharose fractionation of mouse ascites fluid containing anti-tetanus antibody. The tetanus toxoid-coated plates have a shelf life of at least 1 year.     

Immunogenic properties in mice of hexasaccharide from the capsular polysaccharide of Streptococcus pneumoniae type 3.

Hexasaccharide (HS) containing 3 U of cellobiuronic acid was isolated from Streptococcus pneumoniae type 3 capsular polysaccharide S3 and coupled to bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or tetanus toxoid (TT). The immunogenicity of these HS-protein conjugates in BALB/c mice was studied by measuring the production of circulating antibodies and the induction of protective immunity to viable S. pneumoniae type 3. Immunization of BALB/c mice with 0.5 micrograms of S3 resulted in the induction of immunoglobulin M (IgM) antibodies and complete protection against 25 U of a mean lethal dose of S. pneumoniae type 3 for 19 weeks after immunization. BALB/c mice immunized with 100 micrograms of HS9-BSA (containing 12 micrograms of HS) were also protected due to circulating IgM antibodies. Repeated injections with either 100 micrograms of HS9-BSA (three immunizations) or 100 micrograms of HS6-KLH (two immunizations) resulted in high levels of circulating IgG antibodies. These HS-protein conjugates induced complete protection which lasted at least 14 (HS9-BSA), 23 (HS6-KLH), or 8 (HS16-TT) weeks after the last immunization. Protection against viable S. pneumoniae type 3 could be passively transferred to nonimmunized mice by antisera containing IgM or IgG antibodies or both. Sera containing both IgM and IgG antibodies gave better protection than sera containing only IgM antibodies. The specificity of the induced protection was confirmed by challenge with the non-cross-reacting S. pneumoniae type 11.