Induction of antibody responses in mice immunized intranasally with Type I interferon as adjuvant and synergistic effect of chitosan.

Type I IFNs are a range of host-derived molecules with adjuvant potential; they have been used for many years in the treatment of cancer and viral hepatitis. Therefore, the safety of IFNs for human use has been established. In this study, we evaluated the mucosal adjuvanticity of IFN-ß administered intranasally to mice with diphtheria toxoid, and suggested a method to improve its adjuvanticity. When IFN-ß alone was used as a mucosal adjuvant, no clear results were obtained. However, simultaneous administration of IFN-ß and chitosan resulted in an enhancement of the specific serum immunoglobulin G (IgG) and IgA antibody responses, the mucosal IgA antibody response, and antitoxin titers. Furthermore, the intranasal administration of IFN-α alone resulted in a greater increase in antibody titer than IFN-ß, and a synergistic effect with chitosan was also observed. These findings suggest that intranasal administration of chitosan and Type I IFNs may display an effective synergistic mucosal adjuvant activity.

[Diphtheria: a 'forgotten' disease?] / Difterie: een 'vergeten' ziekte?

Respiratory diphtheria is an acute respiratory tract infection. The high mortality rates (5-10%) are related to airway obstruction and local and systemic effects of the diphtheria toxin. Vaccination against diphtheria has been available through the Dutch national vaccination programme since the 1950s. The disease has now become rare as a result of herd immunity by these vaccinations. As a consequence, the disease has largely been forgotten, which can result in it not being recognised and treated in time. In addition, diphtheria antitoxin is not always available. In this article, we are drawing attention to the need for immunisation. We also look back at how diphtheria prevention started in the Netherlands.

Characterization of serum anti-diphtheria antibody activity following administration of equine anti-toxin for suspected diphtheria.

There is a global shortage of equine-derived diphtheria anti-toxin (DAT) for diphtheria treatment. There are few existing data on serum antibody concentrations and neutralizing activity post-treatment to support development of new therapeutics. Antibody concentrations were quantified by ELISA and anti-toxin neutralizing activity by cytotoxicity assay in serum from 4 patients receiving DAT for suspected diphtheria. Using linear mixed effects modeling, estimated mean (SE) half-life was 78.2 (20.0) hours. Maximum serum neutralizing activity ranged from 28.42-38.64 AU/mL with an estimated mean AUC1-72 of 1396.7 (399.3) AU/mL*hr. These data provide a standard of comparison for development of novel anti-toxins to replace DAT.

Postbooster Antibodies from Humans as Source of Diphtheria Antitoxin.

Diphtheria antitoxin for therapeutic use is in limited supply. A potential source might be affinity-purified antibodies originally derived from plasma of adults who received a booster dose of a vaccine containing diphtheria toxoid. These antibodies might be useful for treating even severe cases of diphtheria.

Calibration and commutability assessment of the 1st International Standard for Diphtheria Antitoxin Human.

The 1st International Standard for Diphtheria Antitoxin Human (coded 10/262) was established by the World Health Organization Expert Committee on Biological Standardization in 2012. This paper describes the production, characterization and calibration of the new standard which is intended for use in the standardization of assays used to measure diphtheria antibody responses in human serum. The new standard was calibrated in terms of the International Standard for Diphtheria Antitoxin Equine in an international collaborative study. A total of 8 participants from 8 different countries performed in vivo and/or in vitro toxin neutralization tests and returned data that was used to assign units to the proposed new standard. The new standard has a diphtheria antitoxin potency of 2 IU/ampoule and is predicted to be stable. A follow up study was performed to assess commutability of the new standard. The follow up study was an existing external quality assessment, modified to include the new standard. Results obtained suggest that the new standard is commutable, showing comparable behaviour to native human serum samples in the majority of the assays compared, and is therefore suitable for use as a reference preparation in assays used to measure the level of anti-diphtheria antibodies in human serum.

Report: seroprevalence of corynebacterium diphtheriae among vaccinated population of Rawalpindi/Islamabad, Pakistan.

Diphtheria is a communicable disease of global significance, and its outbreaks have to be reported to the world community under the International Health Regulations (IHR). A pilot seroepidemiological survey was conducted to assess immunity status of diphtheria among healthy individuals of Rawalpindi/Islamabad (Pakistan), who had been administered at least one dose of the vaccine against the disease, as part of childhood vaccination. The study group comprised of 128 healthy subjects, grouped according to the decade representing their age. Antidiphtheria IgG levels were measured by Enzyme Linked Immunosorbent Assay (ELISA) method. The studied sample showed 100% prevalence of diphtheria antitoxin, confirming prior vaccination; however 49.2% exhibited only minimal protection against diphtheria. Full protection was observed in a significantly higher (p=0.013) percentage of males (54.45%) as compared to female subjects (33.33%). Maximum level of serum antibodies were seen in 1-10 year age group (0.195+0.031 IU/mL), which was significantly higher than that recorded in the age group of 11-20 (p=0.024) and above 30 years (p=0.0064). The present results emphasize the need for periodical booster immunization in adolescents and adults, after primary childhood immunization.

Effective humoral immunity against diphtheria and tetanus in patients with systemic lupus erythematosus or myasthenia gravis.

INTRODUCTION: Controversy exists about the effectiveness of vaccine-induced immune response in patients with immunoregulatory disorders. Our aim was to determine the antibody titers to diphtheria and tetanus in patients with either of two autoimmune diseases. METHODS: 279 patients with SLE (205 females, aged 45.0 ± 13.8 years), 158 patients with myasthenia gravis (MG) (101 females, aged 55 ± 18.7 years) and 208 healthy subjects (122 females, aged 48 ± 14.6 years) were enrolled. Serum concentrations of diphtheria-antitoxin-IgG (A-DIPHTH) and tetanus-antitoxoid-IgG (A-TET) were determined with ELISA. RESULTS: Equal proportions of healthy subjects, as well as patients with SLE or MG exhibited proper antibody responses and immune protection against diphtheria and tetanus. In all three test groups, serum concentration of A-DIPHTH decreased significantly (p<0.001) with age throughout the study population, while titers of A-TET dropped only in the elderly (>60-years-old) subjects. There were no significant differences among the groups in the age-related changes of A-TET and A-DIPHTH except that in <40-years-old subjects, A-DIPHTH level was significantly (p=0.029) lower in SLE patients than in controls. CONCLUSIONS: Our findings suggest that the level of vaccine-induced immunity against diphtheria and tetanus infections in patients with SLE or MG is comparable to the healthy population.

[Immunological similarity of diphtheria toxin and EGF receptor].

Cardiomyopathy and neuropathy are the two commonly observed complications in diphtheria patients and in, some instances, individuals vaccinated against diphtheria. The nature of these complications remains not well understood. It was suggested that autoimmunity may play a role in the development of these afflictions. Based on functional similarities between diphtheria toxin (DT) and epidermal growth factor receptor (EGFR), which both can bind to the heparin-binding EGF-like growth factor (HB-EGF) precursors, we suggested that antibodies developed against DT can cross react with EGFR. Here, using serum from healthy donors (n = 10) and diphtheria patients (n = 15), we demonstrated that B-subunit of DT has the antigenic epitopes similar to those of EGFR. Diphtheria toxin as well as EGFR could be recognized by antibodies raised against EGFR and by serum antibodies from diphtheria patients. Moreover serum of diphtheria patients competitively inhibits binding of anti-EGFR antibodies to the receptor. The truncated diphtheria toxin without B-subunit could be detected by serum antibodies of diphtheria patients, but not by anti-EGFR antibodies. Collectively, these studies demonstrate cross-reactivity of antibodies raised against B-subunit of DT and extracellular domain of EGFR and suggest that clinically observed post-diphtheria complications may result from autoimmune inhibition of EGFR function and possible destruction of receptor-positive tissues.

[Comparative efficacy of homologous and heterologous biologicals against diphtheria].

AIM: Comparative assessment of efficacy of homologous and heterologous diphtheria antitoxins on the example of diphtheria intoxication. MATERIALS AND METHODS: Homologous hyperimmune sera were obtained through immunization of rabbits and guinea-pigs with diphtheria toxoid according to schedule. Immune rabbit sera contained 70 - 100 IU/mL of antitoxin antibodies and guinea-pig sera contained 60 - 80 IU/mL. Equine diphtheria antitoxin was used as a heterologous one. Measurement of antitoxin level using experimental animals is based on quantitative assessment of ability of studied sera to neutralize specific dermonecrotic effect of diphtheria toxin. RESULTS: Concentration of antitoxin in blood of different groups of guinea-pigs immunized 2 days earlier with either heterologous equine antitoxin or homologous antitoxin was 0.06 - 0.125. IU/mL. Animals from both groups were completely protected after administration of 5.64 LD50 of toxin. Alongside with it, 50 - 75% of animals which received homologous antitoxin were protected from higher doses of toxin, whereas all animals which received heterologous antitoxin died after administration of higher doses. After administration of identical doses of homologous antitoxin to rabbits its maximal concentration was observed on the next day, was stable up to 5 - 7 days after injection, decreased two-fold to 12th day and did not change further to 15 - 16 days after injection with subsequent another two-fold decrease to 30th day (then was stable for another 5 - 10 days). CONCLUSION: Administration of homologous antitoxin compared to heterologous analogue allows to prolong time of circulation of specific antitoxic antibodies in 3 - 4 times.

[Specific immunity in patients with diphtheria].

AIM: To study features of specific immunity in patients with diphtheria using data from clinic as well as from animal experiments. MATERIALS AND METHODS: Serum samples of 80 patients hospitalized to Infectious Diseases Clinical Hospital No. 2 in Moscow and treated with anti-diphtheria serum (manufactured by "Immunogen" Concern, Stavropol) were studied. Localized diphtheria of the oropharynx was diagnosed in 29 patients, diffused diphtheria--in 8, subtoxic--in 3, grade 1 toxic--in 19, grade 2 toxic--in 12, grade 3 toxic--in 9. Experimental part of the study was performed on outbred rabbits weighted 3-3.5 kg. Level of antitoxin in serum was measured in reaction of passive hemagglutination using commercial antigenic erythrocyte diagnostic kit (manufactured by Mechnikov Research Institute of Vaccines and Sera, Moscow). RESULTS: Intermittent administration of toxin to control rabbits which lack background immunity did not lead to changes in their immune status and after administration of anti-diphtheria antitoxin kinetics of its level in serum was analogous to that observed after administration of antitoxin to intact animals. Highest level of antitoxin (1.0-2.0 IU/ml) was observed 1-3 days after its administration, and to day 13-15 antitoxin was not detected in serum samples. Diphtheria antitoxin in concentration from 0.03 to 40.0 IU/ml was detected in serum samples in 59 of 80 (74%) studied patients. Only in 21 patients (26%) the antitoxin was not detected. CONCLUSION: Presence of antitoxin in serum argue for active immune response to infection and activation of immune memory mechansisms, which allow to predict less severe course of the disease. Absence of antitoxin in serum of patient admitted to hospital points that infectious process is developing on the background of no immunity that predicts the probable development of severe diphtheria.

A review of the international issues surrounding the availability and demand for diphtheria antitoxin for therapeutic use.

Diphtheria treatment requires early administration of diphtheria antitoxin (DAT), an immunoglobulin preparation that neutralises circulating diphtheria toxin. Here, we review issues relating to the supply and use of DAT and assess its availability by means of an international survey. Results showed that several countries do not currently hold DAT stockpiles due to low prevalence, and hence perceived risk of diphtheria, and/or difficulties in obtaining DAT supplies. The potential for importation of cases into any country exists globally, since diphtheria remains endemic in many regions. It is therefore important that DAT be readily available - particularly since waning diphtheria immunity has been observed among adult populations in countries with good vaccination coverage. Options for diphtheria therapy are discussed.

High tetanus and diphtheria antitoxin concentrations in Finnish adults--time for new booster recommendations?

The tetanus and diphtheria vaccination programme in Finland has been running for 50 years. After primary doses, tetanus boosters have been offered to men in military service and decennial boosters recommended for all through the adult life. For 30 years a diphtheria booster was only offered to men in the military service. Not until 1989 diphtheria-tetanus (dT) and diphtheria (d) booster vaccines for adolescence and adults were introduced. In this study serum samples of 990 subjects from 30 years of age, participating in a population survey in 2000-2001, were used to assess the tetanus and diphtheria antitoxin concentrations. More than 70% of the adults up to 50 years of age were fully protected (antitoxin concentrations >0.1 IU mL) against tetanus and diphtheria. Of these adults more that 76% had antitoxin concentrations >1 IU/mL against tetanus, indicating long-term protection but also an increased risk for hyperimmunisation. A comparison of this study and two immunogenicity studies conducted in Finland in 1987-1988 and 1995-1996 shows the impact of an active decennial dT adult booster programme in a country with a high primary tetanus and diphtheria vaccination coverage in infants since the 1950s. Recommendations for limited decennial boosters by increase the time interval between dT boosters up to 20 years as suggested by this study and also studies performed, e.g., in Denmark and Portugal should be considered. Finnish adults born before 1930 should, however, still be vaccinated with decennial boosters, especially against tetanus.

Standardization and validation of Vero cell assay for potency estimation of diphtheria antitoxin serum.

Diphtheria toxin has the capacity to block protein synthesis in cultured mammalian cells, and thus causing cell death. This capacity of diphtheria toxin was utilized for in-vitro neutralization test to determine antibody titer, using Vero cells, which have been found to be susceptible to diphtheria toxin. In the present study, a Vero cell assay was standardized and validated for potency estimation of diphtheria antitoxin serum (DATS). The results obtained by Vero cell assay were compared with in-vivo biological assay. High degree of correlation (+0.98) was found between in-vivo biological assay and in-vitro Vero cell assay. The assay has also been found to be effective in determining the rising antibody titer in the equines inducted in DATS production. The present study indicated that although biological assays hold the key for final potency estimations till date but in the future scenario in-vitro Vero cell assay may be a good alternative to in-vivo biological assay.

[Immunity to diphtheria in population of Rostov-on-Don city and Rostov region in recent years].

AIM: To study the magnitute of antitoxic immunity against diphtheria in different age groups of Rostov-on-Don city and Rostov region population. MATERIALS AND METHODS: Antitoxic immunity against diphtheriawas studied in children 3 - 15 years old (661 persons) and adults 16 - 59 y.o. (893 persons), which were tested during 2003 - 2007 using direct hemagglutination assay manufactured by Mechnikov Scientific Production Association "Biomed" (Moscow). RESULTS: Proportion of children 3 - 15 y.o. seropositive to diphtheria was 98.6 +/- 0.5%. Children aged 3 years and 4 - 6 years were identified as a risk group for diphtheria as they had highest proportions of non-immune persons. Proportion of immune to diphtheria in adults was 94.4 +/- 0.8%. Maximal level of protection was observed in personsaged 16 - 17and 18 - 19 years, in which the proportion of seropositive persons was 99.1 - 100%. Persons > or = 50 years old formed group of risk because persons with low or moderate titers of antitoxin were identified in this age group mostly. CONCLUSION: Obtained results provide evidence on enough high level of protection of Rostov-on-Don cityand Rostov region population against diphtheria. Group of increased risk comprised of persons aged 3 - 6 years and > or = 50 years.

[Observation of immunity level of pertussis-diphtheria-tetanus in healthy people in Hangzhou City during 1995-2006].

OBJECTIVE: To study the pertussis-diphtheria-tetanus immunity level of healthy people in Hangzhou. METHODS: Pertussis antibody, diphtheria antitoxin and tetanus antitoxin were tested. We carried out surveillance in different counties among the people at 2-4, 6-8, 13-15, 25-39 age group during 1995-2006. RESULTS: It showed that the pertussis antibody positive rate of 1942 persons was 93.87%, GMT 1: 1037.36 IU/ml, the antibody of people in countryside for the people at 2-4 age group was lower than that of other groups and the difference was significant compared with 6-8, 13-15 age group (chi2 = 8.80, 6.13, P < 0.01). The diphtheria antitoxin positive rate of 2141 persons was 91.59%, with the GMT was 0.2911 IU/ml. The tetanus antitoxin positive rate of 2141 persons was 72.35%, GMT 0.095 IU/ml. CONCLUSIONS: We need to strengthen the EPI work in rural areas.

[Mechanisms of immune response during diphtheria infection].

State-of-the-art data about mechanisms of immune response against diphtheria are presented. Antitoxic and antibacterial immunity as well as factors of natural resistance are characterized. Allergic and autoimmune reactions during diphtheria and their role in the development of complications are reviewed. Literature data and results of authors' studies in area of immunity against diphtheria are included in the review.

[Study of diphtheria anatoxins in immunochemical and tissue culture assays].

Equine diphtheria antitoxins from different manufacturers were studied. Their immunochemical interaction with diphtheria toxin, toxoid, and antigens of Corynebacterium diphtheriae in ELISA and immunoblotting assays as well as biological activity in CHO cell assay were compared. The discovered differences between antitoxin samples with stated equal activity in IU/ml point to heterogeneity of antigen composition in preparations used for immunization. Mentioned methods allow to standardize antitoxins basing on their biological activity and immunochemical characteristics.

[Study on the immunity of diphtheria in children at county level with different 4-vaccine coverage rates based on the results from "National Immunization Program Review" in 2004].

OBJECTIVE: To investigate the immunity level of diphtheria antibody among children living in the areas where different coverage rates of 4-vaccines stratified by results of national immunization program (NIP) reviewed in 2004. METHODS: According to data from 4-vaccine coverage rates of NIP reviewed in 2004, 3 levels could be set. We randomly selected 2 counties at each level and then 10 villages from each county with 42 children involved who were born between 1992 and 2003. ELISA quantitative method was used to test IgG of diphtheria antitoxin. RESULTS: (1) The positive rate of diphtheria antitoxin was only 49.6% with the highest as 78.1% and lowest as 33.0%. There was a significant decreasing trend of this positive rate with the increase of age. The highest (61.2%) fell in the group that were born in 2003 and the lowest (37.6%) was seen among children born in 1992 to 1995. (2) Geometric mean concentrations (GMCs) was only 0.48 IU/ml with a trend of decrease when age was increasing. There was no GMCs peak seen in children who were at the age of boosting, as expected. (3) Positive rates of children born between 2001 and 2003 were lower than 62% while the diphtheria-pertussis-tetanus (DPT) vaccine coverage rates were all higher than 90%. (4) There was no significant difference of diphtheria antitoxin positive rates between children with eligible routine immunization (58.1%) and those were ineligible (59.6%). CONCLUSION: Other than some specific ones, children from most of the investigated counties had a low level of antibody against diphtheria. The coverage rate of DPT vaccine did not necessarily reflect the immunity against diphtheria, suggesting the increase of immunity against diphtheria an urgent task to be taken care of.

Levels of diphtheria and tetanus specific IgG of Portuguese adult women, before and after vaccination with adult type Td. Duration of immunity following vaccination.

BACKGROUND: The need for tetanus toxoid decennial booster doses has been questioned by some experts. Several counter arguments have been presented, supporting the maintenance of decennial adult booster doses with tetanus and diphtheria toxoids (adult formulation of the vaccine: Td). This study aimed to evaluate the use of Td in Portuguese adult women under routine conditions. For that purpose we selected a group of women 30+ years of age to which vaccination was recommended. We intended to know if pre-vaccination antibody concentrations were associated with factors as age at first and last vaccination, number of doses and time since last revaccination. We also intended to assess the serological efficacy of Td booster. METHODS: Following the Portuguese guidelines 100 women were vaccinated with Td. Antitetanus toxin IgG (ATT IgG) and antidiphtheria toxin IgG (ADT IgG) levels were measured (mIU/ml) in 100 pre-vaccination and 91 post-vaccination sera. Detailed vaccination records were available from 88 participants. RESULTS: Twenty-two women (Group A) began vaccination with DPT/DT in their early childhood and their pre-vaccination ATT IgG levels increased with the number of doses received (p = 0.022) and decreased with time since last vaccination (p = 0.016). Among the 66 women who began vaccination in adolescence and adulthood (Group B), with monovalent TT, ATT IgG levels decreased with age at first dose (p < 0.001) and with time since last vaccination (p = 0.041). In Group A, antidiphtheria toxin IgG kinetics was very similar to that observed for ATT IgG. Among women not vaccinated with diphtheria toxoid, ADT IgG levels decreased with age. Serological response to both components of Td was good but more pronounced for ATT IgG. CONCLUSION: Our study suggests that, to protect against tetanus, there is no need to administer decennial boosters to the Portuguese adults who have complied with the childhood/adolescent schedule (6 doses of tetanus toxoid). The adult booster intervals could be wider, probably of 20 years. This also seems to apply to protection against diphtheria, but issues on the herd immunity and on the circulation of toxigenic strains need to be better understood.