Adjuvant effect of Vernonia amygdalina leaf extract on host immune response to hepatitis B virus subunit vaccine.

Aim of the study: Adjuvants can increase the efficiency and reduce the number of required doses for hepatitis B vaccination. Thus the study was designed to investigate whether V. amygdalina leaf extract may be used as an adjuvant to the conventional hepatitis B surface antigen-based vaccine through humoral response analyses. Methodology: The toxicity/safety margin of V. amygdalina was determined using Lorke's method. Immunization was carried out in mice in two phases, phase 1 employed a 3-times vaccination schedule while phase 2 tested 2-times vaccination schedule. The humoral immune response was determined using ELISA test. The total white blood count, different white blood count, aspartate aminotransferase level, alanine aminotransferase level were determined and the body weight of the mice periodically monitored. Results: Our data show that V. amygdalina was not toxic up to the dose of 5000 mg/kg bodyweight (bw). At a concentration of 250 mg/kg bw as an adjuvant in a three times vaccination schedule, it increased IgM, IgG1 and IgA antibody responses. In a 2-times vaccination schedule, 1000 mg/kg of V. amygdalina as an adjuvant to hepatitis B vaccine was able to elicit effective antibody production (0.174±0.002) significantly (P <0.05) higher than the conventional hepatitis B vaccine group (0.109±0.002) which received 3-times vaccine dose. It equally enhanced innate cell-mediated immune response by increasing total white blood cell, neutrophil and lymphocyte counts. The adjuvant-vaccine combination did not produce side effects as the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were within the normal ranges. The liver excised from the sacrificed mice at the end of the vaccination series showed no sign of congestion, inflammation or colour change. Periodic mice body weight monitoring showed similar growth pattern between the treatment and control groups. Conclusion: Results obtained suggest that V. amygdalina may serve as an effective adjuvant to hepatitis B virus vaccine.

Silica nanoparticles as the adjuvant for the immunisation of mice using hepatitis B core virus-like particles.

Advances in nanotechnology and nanomaterials have facilitated the development of silicon dioxide, or Silica, particles as a promising immunological adjuvant for the generation of novel prophylactic and therapeutic vaccines. In the present study, we have compared the adjuvanting potential of commercially available Silica nanoparticles (initial particles size of 10-20 nm) with that of aluminium hydroxide, or Alum, as well as that of complete and incomplete Freund's adjuvants for the immunisation of BALB/c mice with virus-like particles (VLPs) formed by recombinant full-length Hepatitis B virus core (HBc) protein. The induction of B-cell and T-cell responses was studied after immunisation. Silica nanoparticles were able to adsorb maximally 40% of the added HBc, whereas the adsorption capacity of Alum exceeded 90% at the same VLPs/adjuvant ratio. Both Silica and Alum formed large complexes with HBc VLPs that sedimented rapidly after formulation, as detected by dynamic light scattering, spectrophotometry, and electron microscopy. Both Silica and Alum augmented the humoral response against HBc VLPs to the high anti-HBc level in the case of intraperitoneal immunisation, whereas in subcutaneous immunisation, the Silica-adjuvanted anti-HBc level even exceeded the level adjuvanted by Alum. The adjuvanting of HBc VLPs by Silica resulted in the same typical IgG2a/IgG1 ratios as in the case of the adjuvanting by Alum. The combination of Silica with monophosphoryl lipid A (MPL) led to the same enhancement of the HBc-specific T-cell induction as in the case of the Alum and MPL combination. These findings demonstrate that Silica is not a weaker putative adjuvant than Alum for induction of B-cell and T-cell responses against recombinant HBc VLPs. This finding may have an essential impact on the development of the set of Silica-adjuvanted vaccines based on a long list of HBc-derived virus-like particles as the biological component.

The first five years of universal hepatitis B vaccination in South Africa: evidence for elimination of HBsAg carriage in under 5-year-olds.

South Africa implemented a vaccine against hepatitis B virus (HBV) into the Expanded Programme on Immunisation (EPI) in April 1995. The HBV vaccine is given at 6, 10, and 14 weeks, in parallel with OPV, DTP and Hib vaccines. This study assessed the impact of universal childhood HBV vaccination programme in reducing HBsAg carriage, in the first five years (1995--1999) since its implementation. In parallel, we investigated the current burden of HBV infection in mothers of vaccinees and the adult general population. A total of 598 babies (mean age=23.3 months) who received 3 doses of 1.5 microg/0.5 ml Hepaccine-B (Cheil) were recruited from the Northern Province (one of the nine provinces in South Africa). HBsAg, anti-HBs, anti-HBc, HBeAg and anti-HBe were tested using the IMx or Axsym kits (Abbott Laboratories). PCR assays were performed following established protocols. The overall seroprotection rate (i.e. anti-HBs titre> or =10 mIU/ml) was 86.8% (519/598) in vaccinated babies, while 13.2% had anti-HBs levels<10 mIU/ml. Seroprotection rates and geometric mean titres (GMT) decreased significantly with increasing age, possibly reflecting waning anti-HBs titre over time. Total HBV exposure (positive for either HBsAg, anti-HBs, or anti-HBc) was 31.0% (58/187) in mothers of vaccinees and 40% (72/180) in the adult general population. HBsAg carrier rate was virtually similar in both groups (3.2% in mothers of vaccinees vs. 3.3% in the general population). Against this background, no vaccine failures resulting in HBsAg and HBV DNA positivity were seen in vaccinated babies, including 6 babies born to HBsAg positive carrier mothers (one carrier mother was positive for HBeAg and HBV DNA). However, 0.9% (5/582) babies, aged between 8--11 months, tested positive for anti-HBc, all of whom had anti-HBs titres>10 mIU/ml and were negative for HBV DNA. Anti-HBc positivity was probably maternal in origin, or may represent sub-clinical averted HBV infections. It can be concluded that the HBV vaccine is highly effective within the framework of the South African EPI and already shows a positive impact in the elimination of HBsAg carrier rate in children<5 years.

Hepatitis B: vaccination programmes in Europe--an update.

In the eight years since the Global Advisory Group of the Expanded Program on Immunisation set 1997 as the target for integrating hepatitis B (HB) vaccination into national immunisation programs world-wide, more than 116 countries have included HB vaccine as part of their routine infant or adolescent immunisation programs. Meanwhile, many countries have performed economic evaluation studies, while others have initiated sero-epidemiological studies to generate input data for burden of disease calculation. These studies have indicated that epidemiological and economic arguments cannot be used to delay the implementation of universal hepatitis B vaccination. Some countries have improved their surveillance system and included viral hepatitis in the surveillance programs. Other have put hepatitis B vaccination on the political agenda. By the year 2000, following countries of the WHO European Region (51 countries) have implemented a universal hepatitis B immunisation programme: Andorra, Albania, Austria, Belarus, Belgium, Bulgaria, Estonia, France, Germany, Greece, Italy, Israel, Kazakhstan, Kyrgyzstan, Latvia, Lithuania, Luxembourg, Malta, Moldova, Monaco, Poland, Portugal, parts of the Russian Federation, Romania, Slovakia, Slovenia, San Marino, Spain, Switzerland, Turkey and Uzbekistan. The Netherlands and some other European countries are seriously studying the issues or are making budgetary provisions for introduction of HB vaccine into their vaccination programme. Most of the European countries, which now use the vaccine routinely, have started with adolescent or infant immunisation. Belgium (1999), France (1994) and Italy (1991) have begun with both adolescent and infant HB immunisation. France continues since 1st October 1998 with the infant immunisation programme only. The rewards of effective implementation of the programmes in these countries are becoming apparent; and their success offers an exemplary model for other countries. The deadline was 1997. Globally, work still remains to be done to support and implement interventions that will bring us closer to the WHO goal and to control, eliminate and eradicate hepatitis B in the coming generations at large. If all the 145 million infants born in 1991 had been vaccinated in this way, the number of chronic carriers would have been reduced by 7.5 million, and 1.8 million deaths prevented.

Update on hepatitis B vaccination in Italy 10 years after its implementation.

Since the beginning of the Italian program of immunization against hepatitis B, vaccine has been given to more than 9 million children, with an outstanding record of safety and efficacy. The coverage rate is globally around 94%, with differences between Northern and Southern regions, the latter having the lower acceptance rate. According to the National Surveillance System (SEIEVA), the incidence of acute hepatitis B per10(5) inhabitants declined from 5.4 in 1990 to 2.9 in 1998. The reduction was even greater among 15-24-year-old individuals, where the incidence rate per 10(5) decreased from 17.3 to 4.2 in the same period. In parallel with the decline of hepatitis B, Delta hepatitis has also dropped significantly. We expect that by the year 2003 (12 years after the beginning of the program) this vaccination strategy will have led to the protection of all Italians aged 0-24 years, who are those at the higher risk for acquiring hepatitis B virus (HBV) and for developing the chronic carrier state.

Impact of hepatitis B immunisation as part of the EPI.

Since 1992, hepatitis B vaccine has been an integrated part of Thailand's expanded programme on immunisation (EPI). Based on the data from five representative provinces, we have evaluated its impact on the countrywide prevalence of HBV infection and carrier rate. The population studied comprised 400-488 healthy and immuno-competent, subjects per area. The subjects' ages ranged from 6 months to 18 years. We examined their sera for viral hepatitis markers using commercially available test kits and established the coverage rate of hepatitis B vaccination after its inclusion into the EPI to be 71.2-94.3%. The number of individuals undergoing the complete course of vaccinations had increased four-fold. Consequently, only 0.7% of the children born after the implementation of this the novel EPI strategy were HBV carriers.

Evaluation of the hepatitis B prevention education programme in Poland.

Evaluation of the hepatitis B prevention education programme was performed as a survey including all the coordinators in 25 regions (211 coordinators; half the country). The success of the programme was defined by an objective measure (the ratio of vaccinated children to the total number of children in a region) and a subjective measure (the need to introduce changes in current procedures). The best information was felt to be provided on the subjective area, with financial aspects raising the most doubts. There was a high level of participation in local training by school nurses, but insufficient participation by paediatric nurses and paediatricians. In one-third of the regions schools fulfilled the tasks set them, but in 19% of the regions only a few schools did. Information about vaccination was given in most public children's clinics in 65% of the regions, but there was no activity in 6% of the regions. Recruitment of sponsorship was successful, with only 12% of parents paying the full cost of vaccination. The most important factors contributing to the success of the education programme were the health education and the epidemiology departments of state sanitary inspection, the schools' medical services and paediatric services. The greatest perceived need for change in procedures was in the area of cooperation with local authorities. Only 56% of those surveyed felt the director of the local sanitary station participated to a significant extent, though the programme is very dependent on such participation. Overall, 12% of the regions achieved a ratio of 20 or more between vaccinated and non-vaccinated children, with another 52% reaching a ratio of 5-19.9. The perceived need for changes in cooperation with the Health Service was smaller in regions in which the ratio of vaccinated children was higher. The survey showed a connection between the results of the education programme and the incidence of hepatitis B virus infection.

Hepatitis B school-based vaccination programmes in the USA: a model for hepatitis A and B.

Following the recommendation for routine vaccination against hepatitis B virus for newborns, many states have started school-based catch-up vaccination of 11- to 12-year-olds. Implementation of these programmes requires educational and promotional initiatives to increase awareness among parents, children, teachers, school nurses, school boards and administration. Experience in Framingham, Massachusetts, suggests that over 90% of targeted hepatitis B vaccine coverage can be achieved. Because hepatitis B vaccination targeted at high-risk groups in the USA was largely unsuccessful, this suggests that the initial similar targeted approach with hepatitis A vaccination will also fail. Only about 50% of hepatitis A cases have a known risk factor, and multiple high-risk areas exist throughout the USA. However, the geographical clustering of these high-risk areas and the occurrence of periodic outbreaks, suggest that school-based hepatitis A vaccination programmes may be effective in reducing the risk of infection. A voluntary programme in San Antonio achieved 43% of the targeted coverage in its first year, and a compulsory programme is due to start in Oklahoma. The effectiveness of this programme is not yet known, but future recommendations are likely to include hepatitis A vaccination as a school entry requirement in areas with high incidence of hepatitis A.

Prospects for vaccination against hepatitis A and B in Catalonia (Spain).

Catalonia is in an area of intermediate endemicity for hepatitis A virus (HAV) infection. An Expert Committee has recently proposed the implementation of universal hepatitis A vaccination for 12-year-olds, based on the fact that no risk factors can be identified for hepatitis A in 50% of cases, and also that selective vaccination targeted at high-risk groups has a limited potential to reduce the incidence of hepatitis A. The well-established programme of hepatitis B vaccination of pre-adolescents in Catalonian schools has high levels of vaccination coverage. This will provide a means to introduce hepatitis A vaccination in a cost-effective way in schools, by replacing the single vaccine with the combined hepatitis A and B vaccine. High-risk groups will also continue to be targeted. A pilot programme has commenced in the 1998/1999 school year and will be evaluated after 3 years. If it is successful, it will be extended indefinitely.

From hepatitis B to hepatitis A and B prevention: the Puglia (Italy) experience.

The incidence of hepatitis B virus infection in Italy is 10 per 100, 000 population, with most cases occurring in young adults. Vaccination against hepatitis B has been compulsory since 1991 for all newborns and 12-year-olds. In the Puglia region, this programme has reduced the incidence of hepatitis B from 7.4 per 100,000 population in 1990 to 2.4 per 100,000 population in 1996. The number of notified cases of hepatitis B in Puglia decreased from 212 in 1992 to 73 in 1997. As 50% of these cases occurred in young adults, the main aim of the current vaccination programme is to achieve high coverage rates among teenagers and young adults within the next few years. Although the incidence of hepatitis A is only about 5 per 100, 000 overall in Italy, Puglia is an area of intermediate endemicity with a seroprevalence of antibodies to hepatitis A virus (anti-HAV) of about 40% in 18-year-olds. The incidence of hepatitis A is up to 30 per 100,000 between the periodic outbreaks that occur every 2-4 years. Most notified cases occur in adolescents and young adults. The last outbreak of about 11,000 cases of hepatitis A in the Puglia region occurred in 1996-1997, mainly in the summer months in towns with harbours or near the coast. The most important risk factor was initially consumption of raw seafood, but later was personal contact, probably between children. A vaccination programme against hepatitis A was initiated in Puglia in 1997, aiming to vaccinate all infants of 15-18 months and all 12-year-olds against hepatitis A. Infants receive monovalent hepatitis A vaccine with the first dose of mumps/measles/rubella vaccine. Monovalent hepatitis vaccine can be given with the second and third doses of hepatitis B vaccine in 12-year-olds, but use of combined hepatitis A and B vaccine is recommended to aid compliance and reduce the commitment of physician/nurse time. Vaccination can be performed in school.

Awareness campaigns: experience in Mexico.

The current total of AIDS cases in Mexico is 37,000 of which 86% have occurred in men. The major route of transmission is sexual. The campaign to prevent AIDS has fallen into four phases, and has now been extended to other sexually transmitted diseases, including hepatitis B. The first phase (1985-1989) was based around question and answer brochures, which increased awareness but did not remove misconceptions. A mass media campaign addressed these misconceptions and stressed preventive measures. The campaign was halted by opposition to the promotion of condom use on the grounds that it encouraged promiscuity. The second phase (1989-1992) used more conservative messages, but these were too obscure and failed to reach the target audience. A poster campaign using popular lottery characters was widely accepted. In the third phase (1992-1994), a combination of messages was targeted at different populations, including parents and women, and general public sympathy for social support for people with AIDS was encouraged. In the fourth phase (1996-2000), a mass media campaign was aimed at teenagers, with parents and teachers as support groups. The campaign was widened to include HBV infection, and posters and brochures for teenagers were produced. These are distributed as part of a collaboration with non-governmental organizations providing sex education. The private medical sector is being encouraged to provide facilities for hepatitis B vaccination. So far the campaign has only been established in Mexico City, but it is hoped that this will be extended nationwide. Hepatitis B vaccination has been recently included in the National Immunization Programme for infants in the first year of life and it is officially recommended for at-risk populations.

[The effects of zhuling-duotang, hepatitis B vaccine and bacillus Calmette-Guerin on immunoactivities of peripheral blood mononuclear cells: an in vitro research].

"Zhuling-duotang (a polysaccharide preparation of the Chinese traditional herb medicine polyporusum bellatus) and hepatitis B vaccine (HB vaccine)" and "Persantin and bacillus calmeteguerin (BCG)" have been used to treat chronic hepatitis B. To elucidate their therapeutic mechanism, we studied the effects of zhuling-duotang, HB vaccine and BCG on immunoactivities of nomal human peripheral blood mononuclear cells (PBMCs). Cytotoxicity of PBMCs incubated with such drugs against target cells K562, HepG2 or 2.2.15 was detected by using 3H-TdR release assay. IL-2 and IFN-gamma activities of the supernatant were assayed. Immunofluorescent analysis of molecular CD4, CD8, CD16, IL-2R on the surface of PBMCs were done. The results showed that: (1) Zhuling-duotang and BCG could significantly increase the cytotoxicity of PBMCs. They could induce PBMCs to produce IL-2 but not IFN-gamma. They also could stimulate PBMCs to express IL-2R. Zhuling-duotang and BCG did not affect the percentage of CD4+, CD8+, CD16+ cells among PBMCs. (2) HB vaccine did not significantly increase the cytotoxicity of PBMCs. (3) The cytotoxicity of PBMCs induced by Zhuling-duotang combined with HB vaccine was not significantly higher than that of PBMCs induced by Zhuling-duotang alone.