VALUTAZIONE DELL'EFFICACIA E DELL'IMMUNOGENICITÀ A LUNGO TERMINE DELLA VACCINAZIONE ANTI-EPATITE B IN ITALIA: ATTUALITÀ E PROSPETTIVE FUTURE.

INTRODUCTION Hepatitis B virus (HBV) is a partially double stranded DNA virus of the Hepadnaviridae family which includes related viruses responsible for liver injuries in animals. On the basis of genetic variability, human HBV strains are currently divided into 8 major (A-H) and 2 minor (I-J) genotypes with a different geographical distribution. HBV is mainly carried in blood but also in other body fluids such as saliva, semen and vaginal secretions. It is usually transmitted by vertical route in developing and highly endemic countries and by horizontal route, especially by unsafe sexual contacts and intravenous drug use, in developed countries with low or intermediate endemicity. Nowadays, hepatitis B is a potentially life-threatening liver infection because safe and effective vaccines are available since ‘80s. However, such infection is still a serious public health problem worldwide. It is a leading cause of acute and chronic liver disease. Currently about 240 million people are estimated to be chronically infected with HBV and more than 686,000 individuals die each year due to HBV-related liver complications, including cirrhosis and liver cancer. Vaccination is the most effective and economically favourable measure to control and prevent hepatitis B on global scale. The viral genome (HBV-DNA) consists of 4 partially overlapping Open Reading Frames (ORFs): in particular ORF pre-C/C encodes for the core protein (HBc) and ORF pre-S/S encodes for the enveloped proteins, among which the most represented is the viral surface antigen (HBsAg). Specific antibodies against hepatitis B core protein (anti-HBc) generally indicate a natural exposure to HBV. Instead, HBsAg is the main component of the currently hepatitis B vaccines since it contains the viral antigenic determinant common to all HBV strains, called a determinant, which induces the host immune system to produce specific neutralizing anti-HBs antibodies able to recognized the viral protein HBsAg in its originally conformation, thus conferring protection. HBV strains with mutations within the a determinant can cause breakthrough infections also in immunized subjects. In Italy, hepatitis B vaccination started in 1983 targeted to individuals at increased risk of infection and then became mandatory in 1991 for all infants and, until the end of 2003, also for 12-year-olds. The first hepatitis B vaccines were plasma-derived, soon replaced by monovalent vaccines manufactured by recombinant DNA technology. These vaccines were proven to be highly immunogenic and able to confer seroprotection (anti-HBs concentration ≥10 IU/l after primary immunisation with a 3-dose vaccination series) in over 90-95% of healthy children and adults. However their long-term protection is still a debated issue. Moreover, from the XXIX century combined vaccines started to be used for infant immunisation. In 2000, two hexavalent vaccines (Hexavac, Sanofi Pasteur MSD and Infanrix Hexa, GlaxoSmithKline) were licensed in Europe for primary immunisation of children against diphtheria, tetanus, pertussis, poliomyelitis, hepatitis B and Haemophilus influenzae B. In 2005, the marketing authorization of Hexavac vaccine was suspended by the European Medicines Agency (EMA) because of concerns about the long-term immunogenicity of its hepatitis B component. A surveillance programme has been undertaken to verify the long-term protection against HBV conferred by such hexavalent vaccine. Up till now, the Italian hepatitis B vaccination strategy was a success: in fact it has drastically reduced the incidence of acute infections and the rate of serological HBV markers. However, this policy needs to be constantly monitored to verify its impact in keeping the infection under control. This PhD thesis resumes the results of 3 different studies conducted in order to assess the efficacy of hepatitis B vaccines and vaccination in our country. AIMS Study 1: to assess the persistence of humoral and cellular immunity conferred by monovalent hepatitis B vaccines 18-19 years after primary vaccination (3 doses) in healthy individuals primed as infants (first year of age) or adolescents (12 years old), respectively. Study 2: a) to assess the antibody persistence and immune response to a challenge dose of monovalent DNA recombinant hepatitis B vaccine (HBVAXPRO 5 µg) in healthy pre-adolescents primed 10 years earlier with a primary series (3 doses) of hexavalent vaccines, either Hexavac or Infanrix Hexa; b) to evaluate the safety profile of the monovalent hepatitis B vaccine, HBVAXPRO 5µg/0.5ml, used as challenge in this study. Study 3: to assess the proportion of successfully vaccinated individuals among cases with acute hepatitis B, the proportion of preventable cases if individuals were vaccinated as recommended, and the reasons for immunisation failures in Italy during the 22 years period (1993-2014) after the implementation of universal hepatitis B vaccination. METHODS AND RESULTS Study 1: Eight hundred fourteen subjects were enrolled, 405 teenagers (49.8%) vaccinated as infants and 409 young adults (50.2%) vaccinated as adolescents. All vaccinees were tested for anti-HBs and anti-HBc antibodies (AxSYM AUSAB and AxSYM CORE, Abbott, USA); serum samples found anti-HBc positive were further tested for HBsAg (AxSYM HBsAg V2, Abbott, USA) and HBV-DNA (Cobas TaqMan HBV Test for use with the High Pure System, Roche, USA). Eight young adults were found positive for anti-HBc alone, and were excluded from analysis. Individuals with anti-HBs concentration ≥10 IU/l were considered protected while those with anti-HBs concentration <10 IU/l were offered a booster dose and re-tested 2 weeks later. Overall, 67.9% individuals showed anti-HBs concentrations pre-booster ≥10 IU/l (48.9% among teenagers vs 87.0% among young adults; p<0.001). The antibody geometric mean concentration (GMC) was significantly higher for individuals vaccinated as adolescents than for individuals immunized as infants (102.5 IU/l vs 6.9 IU/l; p<0.001). When boosted, 94.2% of teenagers and 94.7% of young adults showed an anamnestic response (p=n.s.). Post-booster GMCs were similar in both groups (respectively 477.9 IU/l vs 710.0 IU/l, p=n.s.). Subjects with anti-HBs post-booster <10 IU/l were offered to complete a second vaccination course: 54.5% was immunized and all of them showed an antibody protective titre 1-3 months after the third vaccine dose. Study 2: Seven hundred fifty-one pre-adolescents (11-13 years old) immunized as infants with hexavalent vaccines (Hexavac or Infanrix Hexa) were enrolled, and received a challenge dose of monovalent anti-hepatitis B vaccine HBVAXPRO. In total, 749 individuals (99.7%) completed the study (408 in the Hexavac cohort and 341 in the Infanrix Hexa), but 17 were excluded from the further analysis because of deviations in the trial protocol: especially 10 of them (6 in the Hexavac group and 4 in the Infanrix Hexa) were anti-HBc positive without other virological markers (HBsAg and HBV-DNA negative). The remaining 732 subjects, 396 in the Hexavac cohort and 336 in the Infanrix Hexa, were included in the analysis to evaluate the long-term immunogenicity of the two hexavalent vaccines. Ten years after primary vaccination series, only 44.7% had anti-HBs concentration ≥10 IU/l: the frequency of vaccinees with a protective antibody titre was significant lower in the Hexavac cohort than in the Infanrix Hexa (24.0% vs 69.0%; 95%CI: 19.7-28.4 vs 63.8-74.0). One month after the administration of the challenge dose, 89.5% of pre-adolescent included in the study were protected against HBV, but the percentage of seroprotected was still significant lower in the Hexavac group than in the Infanrix Hexa (83.6% vs 96.4%; 95%CI: 79.6-87.1 vs 93.8-98.1). Pre and post-challenge GMCs and post/pre-challenge GMCR of anti-HBs concentrations reflected the same trend. However, considering the proportion of subjects who showed an anamnestic response by achieving post-challenge anti-HBs concentration ≥10 IU/l in the subgroup of individuals with anti-HBs pre-challenge <10 IU/l, there was no statistically significant difference between the two cohorts (78.4% vs 88.5%; 95%CI: 73.6-83.2 vs 80.7-93.9, respectively). All the enrolled pre-adolescents, except one lost during the follow up, were analysed to evaluate the safety profile of the HBVAXPRO 5µg/0.5ml (n=750). Overall 46.1% of subjects experienced at least one adverse event: 40.7% had an injection-site adverse reaction, all reactions were vaccine-correlated and the most frequent was pain (39.3%); instead 11.7% had a systemic adverse event, but only 1.9% was vaccine-induced. Two serious adverse events were reported during the study, none of them was considered by the centre-investigator as related to the HBVAXPRO injection. No death and no subject was withdrawn from the study due to an adverse event. Study 3: Data concerning acute hepatitis B cases reported to the Italian Surveillance System for Acute Viral Hepatitis (SEIEVA) from 1993 to 2014 were analysed. A total of 11,311 cases were enrolled in the study: 10,949 (96.8%) were unvaccinated instead 362 (3.2%) received at least one dose of hepatitis B vaccine. Among the 10,949 unvaccinated cases, 213 (1.9%) escaped mandatory vaccination and 2,821 (25.8%) were not vaccinated despite being at increased risk of infection. Among the latter, the most common risk factors were intravenous drug use, cohabitation with a HBsAg carriers and homosexual/bisexual practices. Almost 37% of the unvaccinated households with HBsAg carriers were aware of their risk. Lack of trust in the vaccination, negative attitude and inaccurate beliefs followed by lack or poor communication and low perceived severity of the disease were the most frequent reasons for vaccine hesitancy. Among the 362 cases vaccinated with at least one vaccine dose, 277 (76.5%) had available immunisation data: 227 (81.9%) were not properly vaccinated, in particular 76 received an uncorrected immunisation series in terms of time and dose of vaccine administration; instead 50 cases (18.1%) received a complete vaccination course according to the correct schedule and before exposure to HBV. Molecular characterisation of 17 of these latter cases by in-house methods showed that 6 were infected with S-gene mutants and 3 of them were also seroprotected (anti-HBs ≥10 IU/l). CONCLUSIONS Study 1: Strong immunological memory persists for at least 18-19 years after immunization of infants or adolescents with a primary course of vaccination with monovalent anti-hepatitis B vaccines. Thus, booster doses are not needed at this time, but additional follow up is required to assess the long-life longevity of protection. Study 2: Over 80% of pre-adolescents showed persistence of the humoral and/or cellular immunity against HBV one month after a challenge dose of HBVAXPRO given 10 years after priming either with Hexavac or Infanrix Hexa. Overall, the Infanrix Hexa group showed a better antibody and immune memory persistence than the Hexavac group. However, a small proportion of subjects in both cohorts seems to have lost the protection against hepatitis B. Further studies will be necessary to clarify this crucial public health issue. Overall, the challenge dose of HBAXPRO was well-tolerated. Study 3: Development of acute hepatitis B in successfully vaccinated individuals is a rare event. Infections caused by vaccine-escape S gene viral mutants are infrequent. Further efforts are essential on one hand to avoid hepatitis B cases when a full and timely vaccination is possible, on the other hand to achieve and maintain a high level of public confidence in the safety and efficacy of hepatitis B vaccination, especially among individuals at increased risk of HBV infection, in order to reach high coverage rates (herd immunity) to protect the single person and especially the entire community.