Anti-tetanus Toxoid Antibody Research Articles

Binding of complement C3 to the heavy chain of anti-tetanus toxoid antibody

Binding of complement C3 to the heavy chain of anti-tetanus toxoid antibody

Recombinant human antibodies: linkage of an Fab fragment from a combinatorial library to an Fc fragment for expression in mammalian cell culture

The combinatorial phage library approach to immunoglobulin repertoire cloning recently made it possible to isolate gene fragments encoding human immunoglobulin G1 Fabs binding with high affinity to specific antigens. Here we describe the construction of genes encoding whole human anti-tetanus toxoid antibodies based on one of these gene fragments and the efficient expression of these constructs by co-transfection of separate heavy and light chain vectors into a Chinese hamster ovary cell line constitutively expressing a viral transactivator protein. This system will be generally useful for the rapid analysis of recombinant antibodies derived from combinatorial libraries.

In vitro production of specific antibody by equine peripheral blood mononuclear cells using tetanus toxoid as a recall antigen

Anti-tetanus toxoid ( tt) antibody (Ig) levels in the supernatant of cultured, pre-immunised equine peripheral blood mononuclear cells ( pbmc) were measured by an indirect enzyme-linked immunoabsorbent assay ( elisa). Optimal anti- tt Ig production occurred at concentrations of stimulating, purified tt of between 0001 and 0·1 μg ml −1, which varied depending on the cell concentration. Optimal anti- tt Ig production was most consistently produced when the cell concentration was 5 × 10 6 ml −1. At this cell concentration maximal anti- tt Ig was induced using 0·1 μg ml −1 tt. At a cell concentration of 5 × 10 6 ml −1 and a tt concentration of 0·1 μg ml −1 anti- TT Ig was first detectable in supernatant on day 5 of stimulation. Maximal levels of anti- tt Ig were present in the supernatant by day 10. No anti- tt Ig was produced in cultures of PBMC from non-immune animals.

Complement deficiency and antibody profile in survivors of meningococcal meningitis due to common serogroups in Italy.

A collaborative survey was carried out in Italy on a group of 59 subjects with a past history of meningococcal meningitis. The aim was to evaluate the prevalence of complement deficiencies, the serogroup of meningococci responsible for the disease and other possible immune abnormalities associated with the infection. Complement analysis allowed the detection of 10 cases (17%) with deficiencies of the terminal components, and in particular six cases of C8 beta, three of C7 and one of C6 defect. Half of the subjects with complement deficiencies had recurrences of meningitis and developed the infection at an older age in comparison with the control group with normal complement activity. The meningococcal C strain was the most diffuse (68%) and infected all the complement-deficient subjects. Evaluation of the antibody response to meningococcal capsular polysaccharides (PS) showed that only 42.5% of the individuals with group C had antibodies as opposed to 83% and 100% of the patients with meningitis due to group A and B, respectively. In all 59 subjects serum Ig as well as IgG subclasses were present, at normal levels for the age. Vaccination of seven out of the 24 subjects without detectable anti-meningococcal PS antibodies with the sole PS A+C induced a normal response in six of them, including a subject with complement defect. In the subject who did not respond to the antigen, the antibodies against the ubiquitous pneumococcal PS type 14 were also lacking, whereas anti-tetanus toxoid (TT) antibodies were normally present. From these data we may conclude the following: (1) the high prevalence (17%) of late complement components defect among survivors of meningococcal meningitis is also confirmed in the Italian population; (2) the serogroup C, responsible for the infections in all the cases with late complement components defect, is highly recirculating in Italy and apparently less immunogenic; (3) specific vaccination with meningococcal PS is a valid prophylaxis in subjects with lack of specific antibodies as well as in subjects with complement defect.

Impact of Haemophilus influenzae Type b Polysaccharide-Tetanus Protein Conjugate Vaccine on Responses to Concurrently Administered Diphtheria-Tetanus-Pertussis Vaccine

To assess whether serum antibody responses to diphtheria-tetanus-pertussis (DTP) vaccine were affected by coadministration of Haemophilus influenzae type b capsular polyribosylribitol phosphate polysaccharide-tetanus protein (PRP-T) conjugate vaccine when given to patients at 2, 4, and 6 months of age. Randomized, double-blind clinical trial. Urban Santiago, Chile. Healthy infants assembled from health centers. Two hundred seventy-eight (74%) of 375 eligible infants participated; 222, who complied with the complete protocol, constituted the primary group under analysis. One of three vaccine regimens was given to study participants at 2, 4, and 6 months of age, either DTP mixed in the same syringe as PRP-T (group 1); DTP and PRP-T given at separate injection sites (group 2); or DTP without PRP-T (group 3). Titers of serum antidiphtheria toxoid, antitetanus toxoid, and pertussis agglutinin antibodies were measured in blood samples taken from patients 2 months after each dose. Serum antidiphtheria toxoid and antitetanus toxoid responses showed no important depressions in the patients receiving PRP-T. In contrast, geometric mean titers (GMTs) of pertussis agglutinins, expressed as reciprocal serum dilutions, after both the second and third doses (GMT2, GMT3) were lowest in group 1 (GMT2 = 89; GMT3 = 1230), intermediate in group 2 (GMT2 = 123; GMT3 = 1995), and highest in group 3 (GMT2 = 210; GMT3 = 3090; P less than .05 for trend group 1 less than group 2 less than group 3 after each dose). Antipertussis toxin and antipertussis filamentous hemagglutinin antibody titers also were depressed in patients who received PRP-T. Follow-up of a subset at 18 months revealed an expected decline of pertussis agglutinin titers to near baseline levels in each group. Concurrent administration of PRP-T vaccine with DTP vaccine, either in the same syringe or at different sites, interfered with antipertussis responses to a primary series of immunizations. Although the clinical significance of this antagonism is uncertain, these data underscore the caution required in decisions to add new vaccines to existing immunization regimens.

Humoral Immunity in Surgical Patients With and Without Trauma

We measured antitetanus toxoid antibody responses after blunt (n = 24) and penetrating (n = 7) trauma and compared them with responses in patients without trauma (n = 55). Patients were defined as anergic or reactive on the basis of delayed type hypersensitivity response. The response to tetanus toxoid vaccination on admission of patients surviving trauma for over 2 weeks was defined as the ratio of day 14 to day 0 serum IgG antitetanus toxoid levels. Antitetanus toxoid responses were normal after both blunt and penetrating trauma. When stratified according to delayed type hypersensitivity responses, patients with trauma showed better antibody responses than patients without trauma. Major infection rates were similar between trauma groups (three of 24 with blunt trauma vs two of seven with penetrating trauma) and independent of delayed type hypersensitivity (two of 20 reactive patients vs three of 11 anergic patients), in contrast to patients without trauma (one of 19 reactive patients vs 15 of 36 anergic patients). We conclude that decreased delayed type hypersensitivity after moderate trauma is temporary, and that this transient immunodeficiency is not as strongly associated with reduced antibody responses and increased risk of infection as anergy in surgical patients without trauma.

Effective production of anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies by serum-free culture of hybridomas

Two hybridoma systems, mouse·human-human (m·h-h) heterohybridoma and human-human (h-h) hybridoma, have been established, and hybridomas secreting anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies (MoAbs), both having a neutralizing activity have been obtained. Cell-line improvement was shown to be an efficient method for improving the productivity in a cell culture process. Two kinds of serum-free media, GFS (a serum substitute)-containing media and polyethylene glycol (PEG)-containing media, have been established to produce human MoAbs. m·h-h Heterohybridomas could be cultivated for a long period by perfusion culture in an agitation vessel, but h-h hybridomas could not. We found that h-h hybridomas show growth-associated antibody production kinetics and established two kinds of long-term cultivation systems: continuous perfusion culture and semicontinuous immobilized perfusion culture. We also scaled up batch culture and short-term perfusion culture to 200-L and 50-L fermentors, respectively. Processes for large-scale purification from the culture supernatants of both GFS- and PEG-containing serum-free media have also been developed.

IgG anti-tetanus toxoid antibody synthesis by human bone marrow. I. Two distinct populations of marrow B cells and functional differences between marrow and peripheral blood B cells

This investigation uses a system for inducing and detecting anti-tetanus toxoid antibody (anti-TT) synthesis to study specific antibody (Ab) synthesis by bone marrow mononuclear cells (MC). We measured the amounts of anti-TT secreted and the number of B cells secreting antibody (Ab). The ELISA plaque detects single B cells secreting specific Ab. The results show that (1) spontaneous anti-TT secretion by MC is higher than spontaneous anti-TT secretion by peripheral blood lymphocytes (PBL) using an ELISA plaque (P less than 0.01); (2) spontaneous anti-TT production by MC correlated with the serum anti-TT titers as measured by an ELISA (r = 0.75, P = 0.005); (3) two types of marrow B cells were identified--one that spontaneously secretes anti-TT and another that produces anti-TT after TT-stimulation; (4) the frequency of anti-TT-secreting B cells is higher in MC than in PBL; (5) the amount of Ab secreted per marrow B cell is not different from that secreted by a peripheral B cell; and (6) marrow B cells could be induced to produce anti-TT in vitro up to 10 months without added cytokines. These results show that bone marrow is a major repository for differentiated B cells that spontaneously produce Abs to maintain circulating Abs titers and for memory B cells that can be induced to produce specific Ab.

Human Natural Killer Clones Enhance in Vitro Antibody Production by Tumour Necrosis Factor Alpha and Gamma Interferon

To determine whether NK cells are involved in the regulation of the antibody response, we studied the effects of human NK clones on B-cell growth and differentiation and the mechanisms involved. We demonstrate that various human NK clones enhance the immunoglobulin production of SAC/rIL-2-activated B cells, e.g. IgG and IgM by up to 230% and anti-tetanus toxoid antibodies by up to 430%. Cell-cell interactions via cell-surface structures, e.g. the CD11a/CD18 molecule, were found to be critical. Subsequently the NK-mediated B-cell regulation involves cytokines, since cell-free supernatants obtained by 48-h cultures of NK clones exerted BCGF and BCDF activity. Neutralization studies and direct determination characterized these cytokines as IFN-gamma and TNF-alpha. The cytokine production of NK clones could be triggered by activated B cells only. Northern blot analysis demonstrated that activated B cells in co-culture with NK clones were able to induce accumulation of mRNA transcripts for IFN-gamma and TNF-alpha in NK cells.

Evidence that FMC7 is a human B cell differentiation antigen

FMC7 is a 105-kDa B cell restricted antigen which is expressed on about 50% of adult human peripheral blood B cells. Seven to ten days following booster immunization with tetanus toxoid, peripheral blood contains a small population of B cell blasts with an increased density of FMC7. The majority of antitetanus toxoid antibody secreting cells (both IgM and IgG) are however found in FMC7 − B cells. These data indicate that upon in vivo B cell activation FMC7 expression initially increases. B cells involved in antibody secretion have lost the FMC7 determinant.

Lymphocyte phenotype does not predict immune function in pediatric patients infected with human immunodeficiency virus type 1

To determine whether assays of lymphocyte phenotype were predictive of antigen-specific immunologic function in children infected with human immunodeficiency virus type 1 (HIV-1), we compared the antigen-specific cellular and humoral functions (tetanus toxoid-induced T lymphocyte blastogenesis and anti-tetanus toxoid antibody) with the patients' T lymphocyte phenotype, determined at the same time. Although both HIV-1-infected patient populations studied (pediatric hemophilia patients and other pediatric patients) had decreases in the values determined by their functional and phenotypic assays, no association between the functional and phenotypic assays was demonstrated. Thus some HIV-1-infected patients with a normal phenotype had no antigen-specific function, whereas other patients with a markedly abnormal T lymphocyte phenotype had normal antigen specific T lymphocyte function. These results indicate that the assessment of HIV-1-infected patients should include assays of antigen-specific immune function in addition to assays of T lymphocyte phenotype.

Immunoregulatory effects of α-endorphin, β-endorphin, methionine-enkephalin, and adrenocorticotropic hormone on anti-tetanus toxoid antibody synthesis by human lymphocytes

This study assesses the effects of adrenocorticotropic hormone (ACTH), α-endorphin (α-endo), β-endorphin (β-endo), and methionine-enkephalin (met-enk) on tetanus toxoidstimulated in vitro specific antibody (anti-TT) synthesis. ACTH and TT-costimulated cultures enhanced antibody (Ab) synthesis over control TT-stimulated cultures at 10 −9 and 10 −11 M and suppressed Ab synthesis at 10 −13 and 10 −17 M. The α-endo and TT-costimulated cultures enhanced Ab synthesis over control cultures at all concentrations tested (10 −7 to 10 −15 M). Cultures costimulated with β-endo and TT enhanced Ab synthesis over control cultures at 10 −11 M and suppressed Ab synthesis at doses above and below 10 −11 M. The met-enk and TT-costimulated cultures produced more Ab than controls at 10 −9 M; and the met-enk and TT-costimulated cultures produced less Ab than controls at other concentrations as low as 10 −17 M and as high as 10 −7 M. The results of this study provide one reason as to why the interactions between the neuroendocrine and immune systems remain unclear.

Expression of Human Anti-Tetanus Toxoid Antibody in Transfected Murine Myeloma Cells

Mouse-human heterohybridoma cell line, GF4/1.1, secretes a human anti-tetanus toxoid (anti-TT) antibody of the γ3 isotype. This line was used as a source of mRNA for the construction of cDNA libraries and the subsequent isolation of the sequences encoding the human heavy and light chains. Expression vectors containing the cDNAs were constructed and introduced into a non-Ig producing, murine hybridoma cell line, together with a dihy-drofolate reductase (dhfr) selectable marker gene. Transfected cells producing human anti-TT antibody were grown in medium with increasing concentrations of the selecting drug (methotrexate). The level of antibody secretion was tested in cells that had adapted to the higher drug concentration and was found to increase in parallel with increases in the steady-state level of Ig mRNA. This increased productivity was not associated with detectable levels of gene amplification. Stable clones were obtained that secrete levels of human antibody in either open suspension cultures or within microcapsules that compare favorably with those of murine hybridomas.

Immunological responses to chronic heat exposure and food restriction in rats

Immunological variables were studied in rats chronically exposed to high environmental temperature (35 degrees C). Responses were compared with those of rats at 25 degrees C both fed ad libitum and pair fed to the decreased intake found in heat-exposed rats. Heat-exposed rats showed slower delayed-type hypersensitivity responses to keyhole limpet hemocyanin. They showed lower counts of peripheral blood total T cells (OX19+) as well as helper T cells (W3/25+) and smaller numbers of splenic T cells. The thymus was decreased in size. Increased levels of serum IgG antitetanus toxoid antibodies were found in heat-exposed rats. [3H]-thymidine incorporation into Concanavalin A (ConA)-stimulated splenic lymphocytes was decreased in pair-fed rats but not significantly altered in heat-exposed rats compared with controls. Heat exposure alters some aspects of both cellular and humoral immune function in a manner different from that induced by comparable food restriction without heat exposure.

Impaired antibody responses in the hyperimmunoglobulin E syndrome

Patients with the hyper-IgE (HIE) syndrome have recurrent bacterial infections with Staphylococcus aureus and other polysaccharide encapsulated organisms. To determine whether an impairment of the antibody response to polysaccharide antigens contributes to infections in this syndrome, we measured serum antibody to the teichoic acid of S. aureus and to the capsular polysaccharide of Haemophilus influenzae type b. Compared to control subjects who had no history of S. aureus infections (N = 14), sera from patients with HIE (N = 9) lacked the expected elevation of serum antibody to teichoic acid ( p > 0.05) and had significantly lower levels of this antibody than sera from 14 patients with atopic dermatitis, complicated by recurrent cutaneous S. aureus infections ( p < 0.01). After immunization with the capsular polysaccharide of Haemophilus influenzae type of vaccine, the antibody response of patients with HIE was significantly impaired compared to that of age-matched control subjects ( p = 0.01). Although patients with HIE syndrome had normal total IgG levels, most patients with HIE but not patients with atopic dermatitis had IgG 2 subclass deficiency. Defective antibody responses in patients with HIE were not restricted to polysaccharide antigens because the serum levels of antitetanus toxoid antibody in these patients were significantly lower than that of control subjects ( p < 0.001). Impaired antigen-specific antibody responses in patients with HIE syndrome may contribute to their increased susceptibility to infection

The effect of the immunomodulator RU 41,740 (biostim) on the specific and nonspecific immunosuppression induced by thermal injury or protein deprivation.

We studied the effect of RU 41,740 (biostim), a primary macrophage stimulator, in the following two immunosuppressive conditions in rats: (1) a 30% full-thickness burn that leads to significant decreases in cell-mediated (delayed-type hypersensitivity [DTH]), humoral (anti-tetanus toxoid antibody production), and nonspecific immunity (Staphylococcus aureus 502a skin abscess) and (2) protein malnutrition using a 2% protein diet for eight weeks. Biostim administered by gastric intubation at dosages of 10 and 50 mg/kg of body weight for five days following thermal trauma did not prevent the DTH suppression induced by the thermal injury, but resulted in a significant dose-related increase in the amount of anti-tetanus toxoid antibody produced. In the malnourished rats given biostim at dosages of 10 and 50 mg/kg of body weight for seven days, there was a significant dose-related increase in the DTH response in the presence of continued protein depletion in these animals, with a modest but significant reduction in the S aureus 502a skin abscess at three days. Antibody production was not affected with this model. Biostim partially overcomes the suppression in humoral immunity following thermal injury, but not cell-mediated or nonspecific immunity. On the other hand, biostim augments both the cell-mediated and nonspecific immune suppression induced by prolonged protein deprivation but does not affect humoral immunity.

Absence of complement fixing antibodies against lipopolysaccharides from Escherichia coli in a subgroup of patients with Crohn's disease.

Complement fixing antibodies against different Escherichia coli lipopolysaccharides were determined in patients with Crohn's disease and in healthy individuals and compared with antitetanus toxoid antibodies. All healthy individuals had antilipopolysaccharide antibodies, 10 of 27 patients with Crohn's disease had no antibodies and six had rapidly changing antibody titres. These abnormalities were found in patients with disease in the colon, with arthropathy and fistula. Antilipid A was found at lower titres in Crohn's disease. Neither antitetanus toxoid antibodies, nor immunoglobulin concentrations were different in patients with or without antilipopolysaccharide antibodies. There was no evidence for circulating immune complexes in patients lacking antilipopolysaccharide antibodies. Certain subgroups of patients with Crohn's disease have altered antibody levels to typical enteral antigens which most likely can be explained by local antibody binding to lipopolysaccharides at inflammatory sites, or by changes in immunoregulation in this disease.

Immunization of mice against tetanus with fragments of tetanus toxin synthesized in Escherichia coli.

Two recombinant plasmids, pTet11 and pTet18, which express nontoxic protein fragments of tetanus toxin in Escherichia coli, were constructed. pTet11 protein (86 kilodaltons) is a fusion between part of the E. coli trpE protein and 441 amino acids of tetanus fragment C, and pTet18 (63 kilodaltons) consists of part of fragment B and all of fragment C of tetanus toxin. The synthesis of these proteins was induced in E. coli cultures, and the proteins were partially purified. Mice were immunized with these proteins, and dose-dependent titers of anti-tetanus toxoid antibodies were obtained. The proteins were able to induce neutralizing antibodies in mice, as demonstrated by the ability of mice immunized with 1 microgram or more of protein to survive challenge with 10 50% lethal doses of tetanus toxin.

Crossreactivity and inheritance of idiotypes restricted to human anti-tetanus toxoid antibodies.

The presence of cross-reactive idiotypes on human IgG antibodies of tetanus toxoid (TT) antigen was assessed by examining the capacity of two anti-idiotypic (ID) antisera raised against IgG (Fab')2 anti-TT (idiotype) from two subjects to bind radiolabeled "idiotype" and to inhibit the binding of radiolabeled TT to IgG from unrelated subjects and from family members of the idiotype donors. Idiotypic crossreactivity with unrelated individuals was infrequent and weak but was frequent and stronger among siblings. The strongest idiotypic crossreactivity was seen between identical twins in studies using four anti-ID raised against the anti-TT idiotypes of two sets of twins. The results of the present study suggest that idiotypic determinants restricted to human anti-TT antibodies are, at least in part, encoded by inherited genes, which are infrequently shared among unrelated individuals.

The effects of aggregated human IgG and IgG-immune complexes on the agglutination of bacteria mediated by non-immunoglobulin salivary agglutinins

Non-immunoglobulin salivary agglutinins (SBA) for bacteria which bind to Streptococcus milleri TJ7 were isolated from parotid saliva and their interactions with human IgG studied. Purified SBA showed a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a molecular weight of approx. 500,000. Heat aggregated human IgG (63 °C, 30 min), but not native IgG, interacted with SBA and thereby interfered with the ability of the SBA to agglutinate Strep. milleri. Immune complexes prepared from tetanus toxoid and isolated human IgG anti-tetanus toxoid antibody also inhibited salivary bacterial agglutination by SBA; antigen (tetanus toxoid) alone or antibody (anti-tetanus toxoid antibody) alone did not have this effect. Direct-binding studies with immobilized SBA on nitrocellulose paper showed that aggregated IgG bound to immobilized SBA and that this binding was inhibited by EDTA. Thus it appears that heat or specific antigen is able to induce an aggregation of IgG which results in the binding of the aggregated form of IgG to SBA.