Induction of antigen-specific isotype switching by in vitro immunization of human naive B lymphocytes

The use of in vitro immunization technology for the generation of human antigen-specific antibodies has essentially resulted in low affinity IgM antibodies, resembling an in vivo primary immune response. We now describe a detailed reproducible protocol for a two-step in vitro immunization, which yields isotype switched, antigen-specific human antibodies. The immunizing antigen was a 30aa synthetic peptide, containing both a B (15aa V3 peptide of the HIV-1) and a T helper cell epitope (15aa peptide from tetanus toxin). The immunization protocol includes: (i) a selection procedure of donors with a memory T cell response against tetatus toxoid; (ii) immunization of mature naive peripheral B lymphocytes in two distinct phases, involving a primary and a secondary step. None of the donors which were examined after primary 7immunization showed at any time an IgG anti-V3 specific antibody response, while all the donors showed an IgM response. After the secondary immunization step, anti-V3 antibodies of both IgM and IgG isotypes were detected. The switch frequency event was high among the tested donors (5/8).     

Analysis of tetanus toxin peptide/DR recognition by human T cell receptors reconstituted into a murine T cell hybridoma

We have previously reported that human T cell receptors (TcR) selected in the class II-restricted (HLA-DRB1*1302) response to a tetanus toxin peptide (tt830-843) frequently used the Vβ2 germ-line segment which paired with several Vα segments and that the putative CDR3 of both α and β chains showed remarkable heterogeneity. To analyze the structural basis for recognition of the tt830-843/DR complex, five of these TcR were reconstituted into a murine T cell hybridoma, 58 α^?β^?, by expressing the human α and β variable regions joined to the mouse α and β constant regions, respectively. The chimeric TcR, expressing the same Vβ germ-line segment (Vβ2), two expressing Vα21.1, twoVα17.1 and one Vα8.1 were shown to have the expected antigen specificity and DR restriction. Two lines of evidence suggested that the putative CDR3, although not conserved in these TcR, played a key role in recognition. First, two TcR with identical V germ-line segments but distinct CDR3 showed large differences in their capacity to react with the ligand. Second, interchanging the α and β chains from tt830-843/DR1302-specific TcR which differed in their CDR3 sequences invariably led to loss of recognition. We also asked whether germ-line Vα17.1 could functionally replace Vα21.1, as they appear to be related in their primary sequence. However, as in the case of CDR3 exchanges, Vα replacement abrogated TcR reactivity. Taken together, these data underline the fine interdependence of the structural components of the TcR binding site in defining a given specificity. Four of the TcR studied displaying promiscuous recognition were also tested against different DR alleles and site-directed mutants. The results of these experiments suggested that, in spite of their structural heterogeneity, anti-tt830-843 TcR may have a similar orientation with respect to the peptide/DR complex. The reconstitution system described herein should represent a valuable tool for detailed studies of human TcR specificity.     

The set of naturally processed peptides displayed by DR molecules is tuned by polymorphism of residue 86

The response to tetanus toxoid (TT) was studied in immune donors that carry two alleles of DR5 that differ only at DRβ residue 86: DRB1*1101 (G86, abbreviated 1101) and DRB1*1104 (V86, abbreviated 1104). A large number of TT-specific T cell clones was isolated and the epitopes recognized in association with 1101 and 1104 were mapped. We found that two epitopes (p2 and p32) can be recognized in association with both 1101 and 1104 while three epitopes (p23, p27 and p30) are recognized in association with 1101, but never in association with 1104. The sets of naturally processed self peptides displayed by 1101 and 1104 were characterized using alloreactive T cell clones. We found that all 1104 alloreactive clones recognize both 1104 and 1101, while ?30% of the alloreactive 1101 clones fail to recognize 1104. Thus it is apparent that both naturally processed TTand self peptides displayed on 1104 molecules represent a fraction of those displayed on 1101 molecules. The mechanism responsible for this differential presentation was investigated by comparing the capacity of 1101 and 1104 antigen-presenting cells to present TTor synthetic peptides to specific T cells and by measuring the binding of these peptides to DR molecules. Three sets of results suggest that V86 acts as a constraint to the binding of naturally processed peptides: (i) all 1104-restricted or alloreactive T cell clones recognize TT- or allo-epitopes presented by 1101 as well, thus ruling out a major effect of V86 as a residue determining T cell restriction specificity; (ii) presentation of naturally processed peptides correlates in general with the capacity of long synthetic peptides to bind to 1101 or 1104 and (iii) while the naturally processed p30 epitope discriminates between 1101 and 1104, a short synthetic peptide binds equally well to and is comparably recognized in association with both 1101 and 1104. Taken together these results suggest that the natural polymorphism at residue 86 might be a molecular switch that finely tunes the complexity of the peptide collection presented on DR molecules.     

Tetanus in patients with chronic wounds – are we aware?

The incidence of tetanus in patients with wounds is unknown; however, recently concern has been raised over the proportion of tetanus cases in which a chronic wound is the portal of entry for Clostridium tetani. Varicose ulcers, dermatosis and necrosed tumours are estimated to be the point of entry for C. tetani spores in 11-14% of three cases. Of diabetic patients in the USA who contracted tetanus, a diabetic foot ulcer was responsible in 25% of cases despite this chronic wounds have yet to be considered as a risk factor for tetanus. An audit was undertaken and a survey devised to form the basis of the data collection to assess if patients with chronic wounds are up-to-date in accordance with the tetanus immunisation programme. Over a 5-day period, the data were prospectively collected and the tetanus status of a 100 patients retrospectively analysed. The status was then compared with general practitioner (GP) records via telephone follow-up. One hundred patients (n = 100) were available in the audit period, with the majority being male (n = 51). The age range was 22-91 years old (median 70 years). Nearly half of the samples (n = 48) were diabetic, with the majority of patients (n = 35) having venous leg ulcers. Only 15% had a biopsy of their wound. The duration of wounds varied from 1 to 480 months. Patients were asked to confirm their tetanus status. Almost half of the patients were unsure of their tetanus status 48% (n = 48), almost a third 30% (n = 30) thought they were not covered and 22% (n = 22) thought they were up-to-date. After confirming with the GP records, the results were as follows: almost half of the patients, 43% (n = 43) were not covered, 33% (n = 33) were up-to-date, 13% had no immunisation records available at the GPs, 10% had no GP contact details and 1% no contact was possible. Currently, tetanus prophylaxis is given based on the vaccination history of the patient but as identified that this can prove to be unreliable. With the burden of chronic wound and ageing population set to increase, levels of protection amplify the risk of tetanus faced by those suffering from chronic wounds. Strict caution should be taken in those patients who were born before the national childhood vaccination programme, implemented in 1961. Moreover, every effort should be made to ensure that such individuals complete their primary course. By ensuring each patient is actively immunised, protection against tetanus, a potential killer, is provided.     

Autonomic impairment in tetanus: delayed baroreflex involvement

Autonomic nervous system impairment plays an important role in the clinical course of tetanus and is thought to be responsible for life-threatening complications. It is believed to be associated with predominance of sympathetic activity. Direct baroreflex involvement has not yet been reported. We hypothesized that impaired baroreflex may contribute to the autonomic cardiovascular dysregulation in tetanus. In a patient with tetanus baroreflex sensitivity was measured on the first 5 consecutive days non-invasively using a Finometer device. Baroreflex gain was calculated as sequential cross-correlation between heart rate and blood pressure. Short-time pulse interval standard deviations (SDNN) were derived. Additionally, heart rate and arterial blood pressure were monitored and recorded continuously. Baroreflex gain values and SDNN were compared to a sex- and age-matched control subject. Compared to the control subject the patient with tetanus initially did not show a significant difference in baroreflex gain values (mean 3.68 vs 3.15, p=0.1). However, in the course of the disease an almost complete baroreflex failure occurred (mean 1.0 vs 3.15 and 0.97 vs 3.15, both p<0.0001). No correlation was found between the dynamics of baroreflex gain values and blood pressure or heart rate variability expressed by standard deviation and variance. All 5 measurements in the tetanus patient showed decreased short-time SDNN when compared to the control subject and healthy standards. In our patient we found baroreflex impairment as a part of complex autonomic dysfunction in tetanus. Furthermore, baroreflex impairment occurred only delayed. Blood pressure instability could not be explained by baroreflex dynamics. We suggest that a shift towards sympathetic activity possibly overruled the effects of decreased baroreflex sensitivity on blood pressure regulation.     

Loss of hilar somatostatin neurons following tetanus toxin-induced seizures

A loss of inhibitory interneurons has been reported in the hippocampus following seizure activity in various animal models of epilepsy and in human epileptic tissue. The question of whether particular populations of inhibitory neurons are similarly affected by the chronic block of inhibition tha tresults after tetanus toxin injections directly into the brain has not previously been addressed. In the present study a unilateral intrahippocampal injection of tetanus toxin into the ventral hippocampus was used to produce a chronic epileptic syndrome characterised by brief seizures that recurred intermittently for 6–8 weeks. The results reveal, for the first time, the morphological changes in somatostatin interneurons following tetanus toxin-induced seizures in the rat. A bilateral short-term increase in immunoreactivity of somatostatin neurons is present 1 week after injection. This is accompanied by an increased intensity of somatostatin-immunoreactive axon terminals in the outer molecular layer of the dentate gyrus, which is more marked on the contralateral side. A chronic and significant loss of somatostatin-immunoreactive neurons was noted in the hilus of the dentate gyrus 2 months later. The significance of the chronic loss of the hilar somatostatin neurons in the control of excitatory activity in the dentate gyrus and whether the acute morphological changes are due to a direct action of the toxin on release mechanisms or as a result of seizure activity are discussed.     

Antitoxin's effect on tetanus toxin induced sphincter pupillae paralysis in the rabbit

Summary The effectiveness of antitoxin in preventing tetanus toxin's blockage of acetylcholine release from the cholinergic nerves of the rabbit iris was tested by injecting purified tetanus toxin into the anterior chamber, and by injecting horse antitoxin at various time intervals either into the anterior chamber, intravenously, or into the anterior chamber and intramuscularly.The results indicate that antitoxin is ineffective in preventing the development of sphincter pupillae paralysis once it is induced by tetanus toxin, prior to appearance of the symptoms.The efficacy of antitoxin treatment depends on the administration of an optimal therapeutic dose.Neither the rate of development, nor the rate of recovery from the maximal pupillary paralysis are altered by antitoxin treatment.The severity, the duration, and the time of complete recovery from the paralysis are directly related to the time antitoxin treatment begins.     

Knowledge of general public and health professionals about tetanus immunization

Objective : To study the awareness among general public and health care providers about tetanus immunization in relation to injuries, and their knowledge about tetanus immunization schedules in children, pregnant females and adults.Methods : It was a cross-sectional study done at a perfect health mela and all the government allopathic health agencies in Delhi.Results : The knowledge of tetanus immunization was poor among general public as well as health care providers. A substantial proportion of them indicated tetanus injection after every injury, which was unwarranted. The knowledge of tetanus immunization schedule for adults was poor among all categories of respondents, though it was comparatively better for pregnant females, but only 75% of doctors and 51.1 % of nursing personnel correctly knew the immunization schedule against tetanus in children.Conclusion : There is a need to upgrade the level of knowledge among health care providers so as to ensure that schedules of tetanus are followed properly and unnecessary repeated immunizations are avoided and the same knowledge is passed on to the general public also.     

Reduced-antigen-content-diphtheria-tetanus-acellular-pertussis and inactivated polio vaccine as a booster for adolescents 10 to 14 years of age

High rates of pertussis disease in adolescents suggest that additional boosting against pertussis would be beneficial. A combined acellular-pertussis-containing booster vaccine (dTpa-IPV; Boostrix Polio, n =440) was compared to separately administered dTpa (Boostrix) and inactivated polio virus (IPV; Imovax Polio^®, n =219), and to DTPa-IPV (Infanrix IPV, n =111) vaccine in a partially blind, randomised controlled trial in 10–14 year olds. One month after vaccination, seroprotection/seropositivity rates for all antigens were similar for all groups. Although pertussis and diphtheria antibody geometric mean antibody concentrations were higher after DTPa-IPV, all subjects had protective antibodies against diphtheria, tetanus and polio, and at least 97% had a vaccine response to pertussis antigens. Reactogenicity of dTpa-IPV was comparable to dTpa + IPV, but dTpa-IPV was generally better tolerated than DTPa-IPV. Conclusion:The combined reduced-antigen-content-diphtheria-tetanus-acellular-pertussis and IPV vaccine is immunogenic and well tolerated when administered to adolescents and could be used to improve the control of pertussis disease in this age group.