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.

Toxigenic Corynebacterium ulcerans isolated from a hunting dog and its diphtheria toxin antibody titer.

Toxigenic Corynebacterium ulcerans is a zoonotic pathogen that produces diphtheria toxin and causes a diphtheria-like illness in humans. The organism is known to infect and circulate among dogs, which can then transmit it to humans. Furthermore, previous studies have found that C. ulcerans is carried by wild animals, including game animals. In the present study, we tested hunting and companion dogs for the presence of toxigenic C. ulcerans and succeeded in isolating the bacterium from a hunting dog. Moreover, several hunting dogs had serum diphtheria antitoxin titers that were higher than the titers required for protection in humans, suggesting a history of exposure to toxigenic Corynebacterium strains. Notably, ribotyping, pulsed-field gel electrophoresis and tox gene sequencing demonstrated that the isolate from the hunting dog clustered with previously characterized C. ulcerans strains isolated from wild animals, as opposed to groups of isolates from humans and companion dogs. Interestingly, the wild animal cluster also contains an isolate from an outdoor breeding dog, which could have formed a bridge between isolates from wild animals and those from companion dogs. The results presented herein provide insight into the mechanism by which the zoonotic pathogen C. ulcerans circulates among wild animals, hunting and companion dogs, and humans.

Comparison of seven commercial enzyme-linked immunosorbent assays for the detection of anti-diphtheria toxin antibodies.

Determination of immune status of patients to diphtheria toxin is based mainly on the results of commercially available ELISA kits. The aim of the present study was to compare the results obtained by ELISAs from seven different manufacturers: Mikrogen, Immunolab, Sekisui Virotech, NovaTec, Virion\Serion, IBL International and Euroimmun. All assays were performed according to the manufacturers' instructions. The concentrations of the anti-diphtheria toxin antibodies in 72 serum samples were calculated on the basis of curves constructed from standards supplied by manufacturers and the new reference material-International Standard for Diphtheria Antitoxin (10/262). The repeatability and reproducibility of all the ELISA kits tested were good. Number of sera with concentrations of the anti-diphtheria toxin antibodies below the WHO-recommended level of protection (0.1 IU/ml) were dependent on the ELISA used: Mikrogen, 20/72 samples (27.7%); Immunolab, 11/72 samples (15.3%); Sekisui Virotech, 0/72 samples (0%); NovaTec 18/72 samples (25.0%); Serion 12/72 samples (16.7%); IBL International, 7/72 samples (9.7 %); and Euroimmun, 17/72 samples (23.6%). However, the results obtained in particular ELISAs, with the exception of Sekisui Virotech, were much more consistent when the concentrations of the anti-diphtheria toxin antibodies in 72 sera measured by using curves constructed from the International Standard 10/262. The data obtained clearly demonstrated that manufacturer-dependent differences between anti-diphtheria IgG ELISA kits exist. The differences in recommendations accepted by the individual manufacturers together with differences shown in our studies in sensitivity greatly affect the clinical interpretation of results.

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.

Maternal immunization with tetanus-diphtheria-pertussis vaccine: effect on maternal and neonatal serum antibody levels.

OBJECTIVE: We sought to determine whether tetanus-diphtheria-pertussis vaccination (Tdap) in pregnancy provides newborns antibodies against pertussis when compared to mothers who did not receive Tdap. STUDY DESIGN: Paired maternal and umbilical cord blood samples were collected at the time of delivery and the serum stored at -86°C. For each paired sample of maternal and cord blood, the medical chart and vaccine history was reviewed to determine whether Tdap was received or not. RESULTS: Newborns born from mothers who received Tdap during pregnancy had significantly higher concentrations of diphtheria antitoxin (P < .001), tetanus antitoxin (P = .004), and antibodies to pertussis toxin (P < .001), filamentous hemagglutinin (P = .002), pertactin (P < .001), and fimbriae 2/3 (P < .001) when compared to newborns from mothers who did not receive Tdap. There was a significant increase in the odds that newborns from mothers who received Tdap during pregnancy have antibodies that may provide protection against diphtheria (P = .0141), pertussis toxin (P < .0001), and fimbriae 2/3 (P = .0146). CONCLUSION: Administering Tdap during pregnancy increases antibody titers against diphtheria and pertussis antigens. Maternal Tdap may prevent neonatal pertussis infection.

Evaluation of INSTAND e.V.'s external proficiency testing program for tetanus and diphtheria antitoxin detection: Lessons for assessing levels of immunoprotection.

OBJECTIVES: The aim of this study was to evaluate the development and status quo of the quality of high throughput in vitro diagnostic testing for tetanus and diphtheria antitoxin antibody (ATX) concentrations based on external quality assessment (EQA) data. METHODS: We analyzed manufacturer-specific data of 22 EQA surveys-each for the detection of tetanus and diphtheria ATX-to check the diagnostic strength of the corresponding in vitro diagnostic systems. RESULTS: While the results were mostly well aligned, individual surveys showed widely dispersed ATX concentrations. The medians of manufacturer collectives deviated from the overall median by up to 8.9-fold in the case of diphtheria ATX and by up to 3.5-fold in the case of tetanus ATX. Such a distribution in the results is particularly critical in the cut-off range for immunity and may lead to an incorrect assessment of vaccination status. CONCLUSION: These results were surprising as there are International Standards for both ATX; however, the results may be linked to the high ATX concentration of the reference material, which deviates considerably from clinically significant concentrations. To increase the accuracy and diagnostic strength of both assays, we recommend a recalibration of the test systems and verification of their traceability to the International Standards.

[Assessment of different regimens of diphtheria serotherapy].

AIM: Efficacy of different treatment regimens with equine diphtheria antitoxin (EDA) was assessed on clinical samples as well as in experiments on animals. MATERIALS AND METHODS: Protective properties and serum concentration kinetics of heterologous antibodies was studied on 12 rabbits and 51 guinea pigs after intramuscular injection of different doses of EDA, in serum samples from 26 patients, which received one intramuscular injection of EDA in various doses as well as in serum samples from 10 patients with diphtheria of different severity, which were treated with EDA in total course dose 100,000-1,500,000 IU. Antitoxin concentration in serum sample was measured with passive hemagglutination assay as well as Jensen toxin neutralization test on rabbits. RESULTS: Experiments on laboratory animals received EDA in dose 150 IU/kg showed high protective effect. For example, rabbits with antitoxin level 1.0-1.25 IU/ ml in serum 24 hours after injection of EDA were 50-250 times resistant to dermonecrotic effect of diphtheria toxin compared with rabbits not received EDA. Guinea pig with antitoxin level 0.5-2.0 IU/ ml in serum 2-48 hours after injection of EDA in dose 150 IU/kg were all protected against 35-50 LD50 of diphtheria toxin. After termination of EDA injection there was sharp decrease of antitoxin level and it was not detected in serum 7 days after. Increase of antitoxin level in serum of animals was not adequate to quantity of injected EDA. Study of serum samples from 26 patients received one intramuscular injection of different doses of EDA showed that doses of antitoxin from 20,000 to 30,000 IU resulted in its presence in serum in concentration 0.5-3.0 IU/ml whereas injection of 50,000 IU or 70,000-100,000 IU resulted in serum concentrations 1.25-10.0 IU/ ml and 2.5-20.0 IU/ml respectively. CONCLUSION: Relatively low doses of EDA provided relatively high level of protection against diphtheria toxin that should be taken into account during treatment of diphtheria patients.

[Immunization of patients with chronic obstructive pulmonary disease with vaccines of Bubo-M and hepatitis B].

AIM: To assess tolerability and immunological activity of Bubo-M vaccine and hepatitis B vaccine in patients with chronic obstructive pulmonary disease (COPD). MATERIALS AND METHODS: Sixty-three patients with moderate and severe COPD aged 35-65 years were immunized against diphtheria, tetanus, and hepatitis B. Bubo-M vaccine as well as vaccine against hepatitis B were used for immunization. Immunologic effect of vaccination was assessed by measurement of serum antibody level to HBsAg as well as to diphtheria and tetanus toxoids. Assessment of antibody level to HBsAg was performed by ELISA, and levels of antibodies to diphtheria and tetanus toxoids--by micromethod in direct hemagglutination assay. Reactogenicity of Bubo-M vaccine was measured according to duration and intensity of local and systemic reactions. RESULTS: The local and systemic reactions were infrequent, serious adverse events after vaccination were not observed. Six months after vaccination, protective antibody titers to hepatitis B, diphtheria and tetanus were determined in all immunized persons--either healthy, or with COPD. During completion of vaccination schedule, significant reduction of acute respiratory infections rate and main disease exacerbations was noted in patients with COPD. CONCLUSION: Good tolerability and high immunogenicity of Bubo-M and hepatitis B vaccines were demonstrated in both groups of vacinees. These vaccines could be recommended for booster vaccination of adults with COPD.

[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.

Seroprotection rate in adults after 1-dose, 2-dose, and 3-dose series of a reduced-diphtheria-tetanus toxoid vaccine (Td) during a diphtheria outbreak in Thailand.

To evaluate seroprotection of different dosing strategies of reduced-diphtheria-tetanus-toxoid vaccine (Td) for adults during a diphtheria outbreak in Thailand, we enrolled 160 healthcare workers and 161 adults aged 20-60 years old and measured diphtheria antitoxin (DAT) level before administration of a Td vaccine. We scheduled a second Td at 4-8 weeks and a third Td at 6-12 months interval. DAT was measured 4 weeks after each dose. DAT levels of ≥0.1 and ≥1 IU/mL were considered as seroprotective and long-term seroprotective. Persons achieving long-term seroprotection were not given a further dose. The baseline seroprotection rate was 32.6%, which increased to 87.1% (95% confidence interval, 83.4-90.8%) after one dose. The seroprotection rate increased slightly with additional doses. The immune response was lowest among persons 30-49 years of age. We suggest 1-dose Td for adults during a diphtheria outbreak, and a 2-dose series being considered for those born before 1980.

Serological immunity to diphtheria and tetanus in healthy adults in Delhi, India.

Widespread childhood immunization with DPT (diphtheria, pertussis and tetanus) has largely eradicated diphtheria and tetanus from many countries. The reduction in the circulation of toxigenic strains has resulted in less natural boosting of adult immunity. As a result, the adult population in countries with high childhood immunization coverage have become susceptible to the disease. The duration of immunity after primary immunization to diphtheria and tetanus is limited and a reduction in immunity is common in adults. With this perspective, the present study was carried out on a random serum sample of 255 healthy individuals aged 20-50 years. The serum samples were tested for immunoglobulin G levels against diphtheria and tetanus by enzyme immuno assays. Fifty-three per cent of adults were unprotected; 22 % were seen to have only a basic protection against diphtheria; 25% were protected against both diseases; and 47% were susceptible to tetanus. The susceptibility was seen to increase with age. To avoid epidemics in the future, immunity must be improved. It is important to treat even the most trivial wound with care and tetanus toxoid immunization. Also, it is necessary to monitor the community for immunity to diphtheria using standard techniques in order to undertake epidemiological surveillances of, and prevention from, these dreadful diseases.

Investigation of immunity level against diphtheria and reinforcement of immunity by booster vaccination for infection control staff in Okayama prefecture.

The prevalence of immunity against diphtheria among Okayama local government staff members involved in diphtheria infection control was measured. Diphtheria booster vaccination was administered to staff members with low antitoxin levels (<0.1 IU/ml) in order to reinforce of immunity. Ninety-one (36.7%) of 248 staff members, 20-69 years of age, had fully protective antitoxin levels (> or =0.1 IU/ml), and the remaining 157 (63.3%) showed levels of <0.1 IU/ml. The rate of full protection was higher in females (44.9%) than in males (22.8%) and was also higher in the diphtheria-pertussis mixed vaccine (born in 1958-1967) and diphtheria-pertussis-tetanus mixed vaccine (born in 1968-) (58.3-61.0%) groups than in diphtheria vaccine (born in 1948-1957) and non-vaccinated (born until 1947) (7.4-18.9%) groups. Though antitoxin levels of 13 (68.4%) out of 19 staff members given booster vaccinations increased to 0.1 IU/ml, 50% of these individuals then showed levels of <0.1 IU/ml after 3 years. Most of the staff members with antitoxin levels of > or =0.1 IU/ml in the non-booster vaccination group maintained their immunity levels for 2-4 years, independent of their history of vaccination. To ensure that staff members of the local government have fully protective antitoxin levels against diphtheria, periodical confirmation of antitoxin levels and booster vaccination should both be systematically carried out.

[Identification of heterologous antitoxin in sera of patients with diphtheria].

Using immobilized diphtheria toxin and peroxidase conjugate of monoclonal antibodies to light chains of equine immunoglobulin a method of quantification of equine antibodies against diphtheria in sera of patients after serotherapy was developed. The sensitivity of indirect enzyme-linked immunosorbent assay was 0.0005 IU/ml, and coefficient of variation did not exceed 10%. It was shown that in patients with toxic diphtheria heterologous antitoxin is eliminated within 4-6 weeks. Level of anti-diphtheria immunoglobulin under the similar severity of disease and dosage of antitoxin can vary in wide ranges and depends from individual's characteristics.

Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test.

A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin) instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test). In this study, the serum titers (values between 1.0 and 19.5 IU) measured by a modified TOBI test (Modi-TOBI test) and toxin neutralization assays were correlated (P < 0.0001). Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r(2) = 0.95, P < 0.0001) and for diphtheria (r(2) = 0.93, P < 0.0001) between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

[The humoral immunity against diphtheria].

Results of the conducted study showed that naturally acquired antibacterial and postvaccinal antitoxic antibodies against diphtheria were found in human blood sera. Challenge of ADT-M toxoid to adults resulted in production of antitoxic as well as antibacterial antibodies in high concentrations. In response to challenge of ADT-M toxoid simultaneously with bacterial vaccine against diphtheria Codivac both antibacterial and antitoxic antibodies were synthesized in blood on optimal physiologic levels. This study revealed dynamics of some specific characteristics of humoral immune response after challenge of two different vaccines against diphtheria--ADT-M toxoid and Codivac vaccine.

Seroprotection against diphtheria and tetanus among Russian and Norwegian teenagers.

Russian children between 7 and 10 years of age have been shown to have significantly higher seroprotection against diphtheria compared to Norwegian children. That was due to a reinforcing dose given on entering school to Russian but not to Norwegian children. The next booster was given at the age of 11-12 years in both countries. We have compared diphtheria and tetanus antitoxin levels among 13- to 14- and 15- to 16-year-old teenagers to see if the difference was maintained among the older age group. Serum samples obtained from 106 Russian and 117 Norwegian teenagers were tested by enzyme immunoassay. The Russian and Norwegian adolescents exhibited adequate rates of protection against diphtheria with similar geometric mean antitoxin concentrations of 1.26 and 1.15 IU/ml, respectively, at 13-14 years, and 0.33 and 0.29 IU/ml at 15-16 years. Differences within the age groups were not significant. However, at 13-14 years the Norwegians were much better protected 2 years after a reinforcing dose of tetanus toxoid than the Russians who had not been boosted for 7 years. At the age of 15-16 the difference diminished and became statistically not significant.

A novel experimental platform for toxigenic and non-toxigenic Corynebacterium ulcerans infection in mice.

Corynebacterium ulcerans is a zoonotic pathogen that can produce diphtheria toxin and causes an illness categorized as diphtheria in the European Union because its clinical appearance is similar to that of diphtheria caused by Corynebacterium diphtheriae. Despite the importance of the pathogen in public health, the organism's mechanism of infection has not been extensively studied, especially in experimental animal models. Therefore in the present study we constructed an intranasal infection system for mice. Mice are insensitive to diphtheria toxin and this has the advantage of excluding the cytotoxic effect of the toxin that might interfere with the analysis of the early stage of infection. Both the toxigenic and non-toxigenic C. ulcerans strains were capable of killing mice within 3 days after inoculation at 10(7) colony-forming units per mouse. In experimentally infected animals, C. ulcerans was detected in the respiratory tract but not in the intestinal tract. The bacterium was also detected in peripheral blood and it disseminated into the lung, kidney and spleen to produce a systemic infection. This experimental infection system provides a platform for analyzing the virulence of C. ulcerans in future studies.

Long-Term Protection against Diphtheria in the Netherlands after 50 Years of Vaccination: Results from a Seroepidemiological Study.

BACKGROUND AND AIMS: To evaluate the National Immunisation Programme (NIP) a population-based cross-sectional seroepidemiological study was performed in the Netherlands. We assessed diphtheria antitoxin levels in the general Dutch population and in low vaccination coverage (LVC) areas where a relatively high proportion of orthodox Protestants live who decline vaccination based on religious grounds. Results were compared with a nationwide seroepidemiological study performed 11 years earlier. METHODS: In 2006/2007 a national serum bank was established. Blood samples were tested for diphtheria antitoxin IgG concentrations using a multiplex immunoassay for 6383 participants from the national sample (NS) and 1518 participants from LVC municipalities. A cut-off above 0.01 international units per ml (IU/ml) was used as minimum protective level. RESULTS: In the NS 91% of the population had antibody levels above 0.01 IU/ml compared to 88% in the 1995/1996 serosurvey (p<0.05). On average, 82% (vs. 78% in the 1995/1996 serosurvey, p<0.05) of individuals from the NS born before introduction of diphtheria vaccination in the NIP and 46% (vs. 37% in the 1995/1996 serosurvey, p = 0.11) of orthodox Protestants living in LVC areas had antibody levels above 0.01 IU/ml. Linear regression analysis among fully immunized individuals (six vaccinations) without evidence of revaccination indicated a continuous decline in antibodies in both serosurveys, but geometric mean antibodies remained well above 0.01 IU/ml in all age groups. CONCLUSIONS: The NIP provides long-term protection against diphtheria, although antibody levels decline after vaccination. As a result of natural waning immunity, a substantial proportion of individuals born before introduction of diphtheria vaccination in the NIP lack adequate levels of diphtheria antibodies. Susceptibility due to lack of vaccination is highest among strictly orthodox Protestants. The potential risk of spread of diphtheria within the geographically clustered orthodox Protestant community after introduction in the Netherlands has not disappeared, despite national long-term high vaccination coverage.