Development of nanoparticle adjuvants to potentiate the immune response against diphtheria toxoid.

BACKGROUND: Over the years, diphtheria was known as contagious fatal infection caused by Corynebacterium diphtheria that affects upper respiratory system. The spread of diphtheria epidemic disease is best prevented by vaccination with diphtheria toxoid vaccine. Aluminum adjuvants were reported to stimulate the immune responses to killed and subunit vaccines. OBJECTIVE: Our study aimed to minimize adjuvant particles size, to gain insight of resulting immunity titer and impact on immune response antibody subtypes. METHODS: Aluminum salts and calcium phosphate adjuvants were prepared, followed by micro/nanoparticle adjuvants preparation. After formulation of diphtheria vaccine from diphtheria toxoid and developed adjuvants, we evaluated efficacy of these prepared vaccines based on their impact on immune response via measuring antibodies titer, antibodies isotyping and cytokines profile in immunized mice. RESULTS: A noteworthy increase in immunological parameters was observed; antibodies titer was higher in serum of mice injected with nanoparticle adjuvants-containing vaccine than mice injected with standard adjuvant-containing vaccine and commercial vaccine. Aluminum compounds adjuvants (nanoparticles and microparticles formulation) and microparticles calcium phosphate adjuvant induce TH2 response, while nanoparticles calcium phosphate and microparticles aluminum compounds adjuvants stimulate TH1 response. CONCLUSIONS: Different treatments to our adjuvant preparations (nanoparticles and microparticles formulation) had a considerable impact on vaccine immunogenicity.

Onjisaponins, from the root of Polygala tenuifolia Willdenow, as effective adjuvants for nasal influenza and diphtheria-pertussis-tetanus vaccines.

Active substances from hot water extracts from 267 different Chinese and Japanese medicinal herbs were screened for mucosal adjuvant activity with influenza HA vaccine in mice. The extract from the root of Polygala tenuifolia was found to contain potent mucosal adjuvant activity. The active substances were purified and identified as onjisaponins A, E, F, and G. When each onjisaponin (10 microg) was intranasally (i.n.) inoculated with influenza vaccine (10 microg) in mice, serum hemagglutination-inhibiting (HI) antibody titers increased 3-14 times over control mice administered vaccine alone after 4 weeks. When each onjisaponin (10 microg) was i.n. inoculated with the vaccine (10 microg) followed by i.n. vaccination of the vaccine alone after 3 weeks, serum HI antibody titers increased 27-50 fold over those mice given i.n. vaccinations without onjisaponins. These same conditions also significantly increased nasal anti-influenza virus IgA antibody titers. Two inoculations with onjisaponin F (1 microg) and influenza HA vaccine (1 microg) at 3 weeks intervals, significantly increased serum HI antibody and nasal anti-influenza virus IgA and IgG antibody titers after only 1 week over mice given HA vaccine alone after the secondary vaccination. Intranasal vaccination with onjisaponin F inhibited proliferation of mouse adapted influenza virus A/PR/8/34 in bronchoalveolar lavages of infected mice. Separate intranasal vaccinations with onjisaponins A, E, F, and G (10 microg) each and diphtheria-pertussis-tetanus (DPT) vaccine (10 microg) of mice followed by i.n. vaccination with DPT vaccine alone after 4 weeks showed significant increases in serum IgG and nasal IgA antibody titers after 2 weeks following secondary vaccination over mice vaccinated with DPT vaccine alone. All onjisaponins showed little hemolytic activity at concentrations up to 100 microg/ml. The results of this study suggest that onjisaponins may provide safe and potent adjuvants for intranasal inoculation of influenza HA and DPT vaccines.

Diphtheria in the Russian Federation in the 1990s.

A resurgence of diphtheria spread throughout the Russian Federation in the early 1990s; diphtheria had been well controlled, but circulation of toxigenic strains of Corynebacterium diphtheriae had persisted since the implementation of universal childhood vaccination in the late 1950s. More than 115,000 cases and 3,000 deaths were reported from 1990 to 1997, and, in contrast to the situation in the prevaccine era, most of the cases and deaths occurred among adults. Contributing factors included the accumulation of susceptible individuals among both adults and children and probably the introduction of new strains of C. diphtheriae. Vaccine quality, vaccine supply, or access to vaccine providers did not significantly contribute to the epidemic. Mass vaccination of adults and improved childhood immunization controlled the epidemic. High levels of population immunity, especially among children, will be needed to prevent and control similar outbreaks in the future.

Epidemic diphtheria in Ukraine, 1991-1997.

In 1991, Ukraine experienced a return of epidemic diphtheria after decades of control that had resulted in <40 sporadic cases reported every year. Increased incidence was first recorded in Kiev, Lviv, and Odessa. By 1993, the epidemic had spread to >50% of the oblasts (provinces) in the country, and by 1995, all regions were affected. In 1995, at the peak of the epidemic, >5,000 cases and >200 deaths were reported. As in Russia, >80% of these cases were diagnosed in persons 16-59 years old. In 1993, the government of Ukraine initiated a program of increased immunization among children and at-risk adults, and by 1995, a mass immunization strategy was adopted in an effort to arrest the epidemic, which was increasing exponentially. In 1996, the number of cases started to decrease, and data from 1998 indicate that the downward trend has continued. It is likely that the diphtheria epidemic in Ukraine started among children, who had been left vulnerable due to inadequate childhood immunizations, and then quickly spread to inadequately protected adults.

Epidemic diphtheria in Belarus, 1992-1997.

In 1990, epidemic diphtheria reemerged in Russia and spread to Belarus in 1992, when 66 cases were reported. Diphtheria cases doubled each year in 1993 and 1994 and peaked in 1995, when 322 cases were reported. Intensified routine immunization of young children and mass vaccination of older children and selected groups of adults were conducted in 1995 and were followed by mass vaccination campaigns targeting all adults in 1996. By the end of 1996, full immunization of >95% of children and coverage of>87% of adults with >/=1 dose resulted in a rapid decline in diphtheria cases. In 1998, only 36 cases of diphtheria were reported. More than 70% of the 965 cases and 26 fatalities reported during 1990-1998 occurred among persons >14 years of age. High levels of immunity among the entire population are needed for rapid control of diphtheria epidemics in the vaccine era.

Diphtheria in Lithuania, 1986-1996.

Diphtheria reappeared in Lithuania in 1986 and rose to epidemic levels by 1992. Between 1991 and 1996, 110 cases of diphtheria were registered, with an incidence of 0.03-1.15/100,000 population. Most cases (84%) and all 17 deaths occurred among persons >/=15 years, most of whom had never been vaccinated. Persons 40-49 years old had the highest average annual age-specific morbidity (1.70/100,000) and mortality (0.53/100,000) rates. Low levels of immunity among individuals 40-49 years old and migration to epidemic areas in Russia and Belarus contributed to the epidemic's occurrence. Between 1991 and 1995, toxigenic Corynebacterium diphtheriae strains were isolated from 84 of all registered patients (76%), and nontoxigenic strains were isolated from 13 (12%). By 1996, two mass vaccination campaigns, which provided one dose of vaccine to individuals 25-30 years old and three doses of vaccine to persons 31-60 years old, helped reduce the number of cases. The first campaign achieved 69% coverage; the second achieved 48% coverage.

Diphtheria in Latvia, 1986-1996.

After nearly two decades without a diphtheria case in Latvia, the disease reappeared in 1986. From 1990 to 1996, case counts were highest among adults 40-49 years of age, school-aged children, and adolescents. Nonetheless, the average annualized incidence of disease was highest among infants and preschoolers. In August 1995, mass vaccination efforts began to provide adults 25-60 years of age with at least one dose of vaccine. By the end of the year, a 77% coverage rate was achieved, resulting in a decrease of reported diphtheria cases by 1996. From February to September 1997, special outreach efforts were focused on hard-to-reach populations; as a result, by June 1997, 55% of adults had received three doses of vaccine. While decreases in the incidence of and morbidity from diphtheria have occurred, additional efforts still need to be concentrated on improving vaccination coverage in adults and children <2 years of age and in reducing mortality from diphtheria.

Diphtheria in Estonia, 1991-1996.

Clinical diphtheria reappeared in Estonia in 1991. Between 1991 and 1996, 61 cases and 5 deaths occurred; 19 cases were among children 5-9 years of age, and 11 were among persons 40-49 years of age. From 1993-1995, vaccine supplies donated by Finland were used in vaccination programs. In 1995, the International Federation of Red Cross and Red Crescent Societies and the Estonian Red Cross launched a mass vaccination campaign targeting the adult population. By the end of 1997, it was estimated that 46% of adults had received at least one dose of vaccine. Although the vaccination campaigns did not target the pediatric population, vaccination coverage in school-aged children remained high due to continuing routine vaccination programs. The reappearance and epidemic of clinical diphtheria cases and the mass vaccination campaign efforts demonstrated that preventive measures are important and must be maintained in order to keep diphtheria under control.

Diphtheria epidemic in the Republic of Georgia, 1993-1997.

Epidemic diphtheria reemerged in the republic of Georgia in 1993. From 1993 to 1997, 1405 cases were reported (28 in 1993, 312 in 1994, 429 in 1995, 348 in 1996, and 288 in 1997), with a cumulative incidence of 25.8/100,000 and a case fatality ratio of 9.5%. During 1993-1997, 53% of the diphtheria cases occurred among persons >/=15 years of age. Unvaccinated patients were more likely to have toxic forms (relative risk=2.24; 95% confidence interval=1.69-2.96) or to die of diphtheria (relative risk=2.24; 95% confidence interval=1. 36-3.68) than those who had received at least one dose of diphtheria toxoid. Improvement in routine childhood vaccination coverage and implementation of mass adult vaccination campaigns have been critical to bringing the epidemic under control. By mid-1998, the overall diphtheria situation in Georgia appeared to have been controlled. Only 53 cases were reported from January to June 1998, representing a 64% decrease from the 148 cases during the corresponding period in 1997.

Epidemic diphtheria in the Kyrgyz Republic, 1994-1998.

The Kyrgyz Republic experienced a widespread diphtheria epidemic during 1994-1998. National diphtheria surveillance and vaccination coverage information were used to describe the course of the epidemic. The epidemic began in August 1994, reached a peak in 1995 with 704 cases (incidence rate: 15.4/100,000 population) and 30 deaths, and declined to an incidence rate of 4.0/100,000 during the first 8 months of 1998. Age-specific incidence was highest in 1995 among persons 15-19 and 20-29 years old. Three rounds of mass vaccination with tetanus and diphtheria toxoids for adult use (Td) were conducted; reported coverage was 69% in 1995 and >95% in 1996 and 1997. Reported routine vaccination coverage with three doses of diphtheria toxoid by age 12 months increased from 62% in 1989 to 98% in 1997. Mass vaccination of the adult population with Td and improvements in childhood vaccination coverage played a major role in controlling the epidemic.

Diphtheria epidemic in the Republic of Uzbekistan, 1993-1996.

The Republic of Uzbekistan, like the other Newly Independent States in the 1990s, experienced epidemic diphtheria during the 1990s. The outbreak in Uzbekistan began in 1993 in southern regions that bordered areas of Tajikistan that were experiencing a very intense diphtheria epidemic. However, the Uzbek epidemic rapidly spread and threatened to involve the entire country. From 1993-1996, 1169 cases of diphtheria were reported, compared with 58 in 1990-1992. Unvaccinated or only partially vaccinated cases were more likely to have clinically severe forms of diphtheria than those who were fully vaccinated. Strong epidemiologic links with the Tajik diphtheria epidemic and the predominance of mitis biotype strains of Corynebacterium diphtheriae in Uzbekistan make it likely that the Uzbek outbreak arose independently of the predominantly biotype gravis epidemic that began in Russia. The epidemic appeared to be due to low population immunity and the large-scale reintroduction of toxigenic strains of C. diphtheriae. Several mass vaccination campaigns and general enhancement of routine immunization procedures led to control of the epidemic in 1996.

[The effect of immunomodulators on the development of antibodies to the diphtheria antigen in mice vaccinated with the DTP adsorbed vaccine]. / Vliianie immunomoduliatorov na vyrabotku antitel k difteriinomu antigenu u myshei, vaktsinirovannykh AKDS-vaktsinoi.

An animal model for the study of the influence of immunomodulators on the development and preservation of postvaccinal antidiphtheria immunity was experimentally selected and the corresponding study was carried out. In this work the following immunomodulators were used: dibasol, prodigiosan, splenin, thymalin, reaferon, tactivin, methyluracyl. The study revealed that by day 120 of observation all immunomodulators stimulated the production of antibodies in higher titers than adsorbed DPT vaccine, introduced without immunomodulators. The most effective action was exhibited by splenin and prodigiosan (injected subcutaneously), dibasol and methyluracyl (administered orally). Two latter immunomodulators, if introduced prior to immunization, are the most promising preparations to be used in practical immunoprophylaxis.

[The immunomodulating properties of a diphtheria bacterial vaccine in in vitro experiments]. / Immunomoduliruiushchie svoistva difteriinoi bakterial'noi vaktsiny v opytakh in vitro.

In this investigation lymphocytes sensitized with Corynebacterium diphtheriae antigens obtained from carriers and convalescents were used. The new diphtheria bacterial vaccine Codivac, in contrast to other comparable preparations (diphtheria toxoid, staphylococcal toxoid, staphylococcal vaccine, levamisole), was found to produce a more direct effect by modulating the levels of T-lymphocytes, depending on their initial levels in the patient. Codivac, together with other preparations, can be used for the study of the problems of immunostimulation and immunocorrective therapy.

Effect of different concentrations of diphtheria toxoid & aluminum phosphate on potency of diphtheria component in DPT vaccines.

An effort was made to determine the optimum concentration of diphtheria toxoid in combination with aluminum phosphate gel in DPT vaccines which may give a safe, potent and economical preparation. The effect of four different concentrations of aluminum phosphate and three different antigenic concentrations of diphtheria toxoid on potency of diphtheria component in DPT vaccine was assessed. A gradual increase in potency was seen with increase in toxoid concentration and a gradual decrease in potency with the increase in aluminum phosphate content. Vaccines made with minimum quantities of toxoid (30 Lf/ml) and aluminum phosphate (3 mg/ml) were found to be highly satisfactory. Vaccines prepared with high antigenic purity toxoid have better potency, as compared to those prepared with a relatively low antigenic purity toxoid.