Hepatitis B Vaccination: A Historical Overview with a Focus on the Italian Achievements.

Vaccination is the most effective way to control and prevent acute and chronic hepatitis B, including cirrhosis and HCC, on a global scale. According to WHO recommendations, 190 countries in the world have introduced hepatitis B vaccination into their national childhood immunization programs with an excellent profile of safety, immunogenicity, and effectiveness. Following vaccination, seroprotection rates are close to 100% in healthy children and over 95% in healthy adults. Persistence of anti-HBs is related to the antibody peak achieved after vaccination. The peak is higher the longer the antibody duration is. Loss of anti-HBs does not necessarily mean loss of immunity since most vaccinated individuals retain immune memory for HBsAg and rapidly develop strong anamnestic responses when boosted. Evidence indicates that the duration of protection can persist for at least 35 years after priming. Hence, booster doses of vaccines are currently not recommended to sustain long-term immunity in healthy vaccinated individuals. In Italy, vaccination against hepatitis B is met with success. In 2020, Italy became one of the first countries in Europe to be validated for achieving the WHO regional hepatitis B control targets.

Solar radiation drives methane emissions from the shoots of Scots pine.

Plants are recognized as sources of aerobically produced methane (CH4 ), but the seasonality, environmental drivers and significance of CH4 emissions from the canopies of evergreen boreal trees remain poorly understood. We measured the CH4 fluxes from the shoots of Pinus sylvestris (Scots pine) and Picea abies (Norway spruce) saplings in a static, non-steady-state chamber setup to investigate if the shoots of boreal conifers are a source of CH4 during spring. We found that the shoots of Scots pine emitted CH4 and these emissions correlated with the photosynthetically active radiation. For Norway spruce, the evidence for CH4 emissions from the shoots was inconclusive. Our study shows that the canopies of evergreen boreal trees are a potential source of CH4 in the spring and that these emissions are driven by a temperature-by-light interaction effect of solar radiation either directly or indirectly through its effects on tree physiological processes.

Key Role of Sequencing to Trace Hepatitis A Viruses Circulating in Italy During a Large Multi-Country European Foodborne Outbreak in 2013.

BACKGROUND: Foodborne Hepatitis A Virus (HAV) outbreaks are being recognized as an emerging public health problem in industrialized countries. In 2013 three foodborne HAV outbreaks occurred in Europe and one in USA. During the largest of the three European outbreaks, most cases occurred in Italy (>1,200 cases as of March 31, 2014). A national Task Force was established at the beginning of the outbreak by the Ministry of Health. Mixed frozen berries were early demonstrated to be the source of infection by the identity of viral sequences in patients and in food. In the present study the molecular characterization of HAV isolates from 355 Italian cases is reported. METHODS: Molecular characterization was carried out by PCR/sequencing (VP1/2A region), comparison with reference strains and phylogenetic analysis. RESULTS: A unique strain was responsible for most characterized cases (235/355, 66.1%). Molecular data had a key role in tracing this outbreak, allowing 110 out of the 235 outbreak cases (46.8%) to be recognized in absence of any other link. The data also showed background circulation of further unrelated strains, both autochthonous and travel related, whose sequence comparison highlighted minor outbreaks and small clusters, most of them unrecognized on the basis of epidemiological data. Phylogenetic analysis showed most isolates from travel related cases clustering with reference strains originating from the same geographical area of travel. CONCLUSIONS: In conclusion, the study documents, in a real outbreak context, the crucial role of molecular analysis in investigating an old but re-emerging pathogen. Improving the molecular knowledge of HAV strains, both autochthonous and circulating in countries from which potentially contaminated foods are imported, will become increasingly important to control outbreaks by supporting trace back activities, aiming to identify the geographical source(s) of contaminated food, as well as public health interventions.

Hepatitis B vaccination.

Hepatitis B virus is a worldwide leading cause of acute and chronic liver disease including cirrhosis and hepatocellular carcinoma. Effective vaccines have been available since the early '80s and vaccination has proved highly successful in reducing the disease burden, the development of the carrier state and the HB-related morbidity and mortality in the countries where vaccination has been implemented.   Neutralizing (protective) antibodies (anti-HBs) induced by vaccination are targeted largely towards the amino acid hydrophilic region, referred to as the common a determinant which is present on the outer protein coat or surface antigen (HBsAg), spanning amino acids 124-149. This provides protection against all HBV genotypes (from A to H) and is responsible for the broad immunity afforded by hepatitis B vaccination. Thus, alterations of residues within this region of the surface antigen may determine conformational changes that can allow replication of the mutated HBV in vaccinated people. An important mutation in the surface antigen region was identified in Italy some 25 years ago in infants born to HBsAg carrier mothers who developed breakthrough infections despite having received HBIG and vaccine at birth. This virus had a point mutation from guanosine to adenosine at nucleotide position 587, resulting in aa substitution from glycine (G) to arginine (R) at position 145 in the a determinant. Since the G145R substitution alters the projecting loop (aa 139-147) of the a determinant, the neutralizing antibodies induced by vaccination are no longer able to recognize the mutated epitope. Beside G145R, other S-gene mutations potentially able to evade neutralizing anti-HBs and infect vaccinated people have been described worldwide. In addition, the emergence of Pol mutants associated with resistance to treatment with nucleos(t)ide analogues can select viruses with crucial changes in the overlapping S-gene, potentially able to alter the S protein immunoreactivity. Thus such mutants have the potential to infect both naïve and immunized people, negatively affecting the efficacy of both the antiviral treatment and the vaccination programs. Despite concern, at present the overall impact of vaccine escapes mutants seems to be low and they do not pose a public health threat or a need to modify the established hepatitis B vaccination programs. The development of novel NAs with a high barrier to resistance is warranted.

Occupational exposure to zoonotic agents among agricultural workers in Lombardy Region, northern Italy.

OBJECTIVES: This study was conducted in Northern Italy with the aim of defining the risk of agricultural workers' contact with biological agents through the determination of serum antibodies against selected zoonotic agents. Immunity against tetanus was also investigated. METHODS: Two groups of agricultural workers consisting of 153 animal breeders (exposed) and 46 non- breeders (controls) were included in the study. In a first group of 103 workers (89 exposed and 14 controls) the serum concentrations of antibodies against Hepatitis E Virus (HEV) were measured, whereas in the second group of 96 workers (64 exposed and 32 controls) the serum concentrations of antibodies against Leptospira spp., Coxiella burnetii, Borrelia burgdorferi, Brucella spp. and Salmonella spp. were addressed. Imunization against tretanus was also studied in this group. RESULTS: Animal breeders showed higher rates of IgG antibodies against Coxiella burnetii (50% vs. 31.2%), and Leptospira spp. (59.4% vs. 43.7%). Results of logistic regression analysis revealed that breeder workers showed a tendency to have higher prevalence of positivity for antibodies to Leptospira spp.and Coxiella burnetii than non-breeders (ORs ~ 3). Only one exposed subject showed antibodies against hepatitis E (none in controls), but when tested with another commercially available kit the percentage of anti HEV IgG positive subjects increased to 22.3% in the exposed, while none of the controls showed positive. None of the subjects showed antibodies against Salmonella spp. and Brucella spp. Italians and other European workers have better protection against tetanus (91%) compared to non-EU workers (81%). CONCLUSIONS: The higher frequency of the presence of serum antibodies to zoonotic agents (e.g. Leptospira spp. and Coxiella burnetii) in animal breeders suggests that they are more exposed to biological agents than workers not involved in animal breeding activities. The risk of contact with HEV deserves further studies because the adoption of different assays can result in significantly different results. The promotion of immunization of agricultural workers might be a priority, in particular for migrants.

Switched memory B cells maintain specific memory independently of serum antibodies: the hepatitis B example.

The immunogenicity of a vaccine is conventionally measured through the level of serum Abs early after immunization, but to ensure protection specific Abs should be maintained long after primary vaccination. For hepatitis B, protective levels often decline over time, but breakthrough infections do not seem to occur. The aim of this study was to demonstrate whether, after hepatitis B vaccination, B-cell memory persists even when serum Abs decline. We compared the frequency of anti-hepatitis-specific memory B cells that remain in the blood of 99 children five years after priming with Infanrix -hexa (GlaxoSmithKline) (n=34) or with Hexavac (Sanofi Pasteur MSD) (n=65). These two vaccines differ in their ability to generate protective levels of IgG. Children with serum Abs under the protective level, <10 mIU/mL, received a booster dose of hepatitis B vaccine, and memory B cells and serum Abs were measured 2 wk later. We found that specific memory B cells had a similar frequency in all children independently of primary vaccine. Booster injection resulted in the increase of memory B cell frequencies (from 11.3 in 10(6) cells to 28.2 in 10(6) cells, p<0.01) and serum Abs (geometric mean concentration, GMC from 2.9 to 284 mIU/mL), demonstrating that circulating memory B cells effectively respond to Ag challenge even when specific Abs fall under the protective threshold.

The worldwide impact of vaccination on the control and protection of viral hepatitis B.

Viral hepatitis B is a leading cause of acute and chronic liver disease worldwide, including cirrhosis and hepatocellular carcinoma. Vaccination is the most effective measure for controlling and preventing hepatitis B and its severe long-term sequelae. According to the World Health Organization (WHO), by the end of 2008 177 countries had introduced hepatitis B vaccination into their national routine neonatal, infant and/or adolescent immunisation programmes, and Italy was one of the first countries to implement a universal strategy of hepatitis B vaccination. The implementation of such vaccination programmes has globally resulted in a marked decrease in disease burden, in the carrier rate and in hepatitis B-related morbidity and mortality. Despite this success, work remains to be done to fully achieve the WHO goal of control of hepatitis B and HBV-related diseases on a global scale.

The global impact of vaccination against hepatitis B: a historical overview.

Hepatitis B virus (HBV) infection is a world wide public health problem of major concern. HBV infection may lead to chronic liver disease, including cirrhosis and hepatocellular carcinoma (HCC). Vaccination is the most effective measure to control and prevent hepatitis B and its long-term serious sequelae on global scale, both in terms of cost-effectiveness and benefit-cost ratios. According to the WHO recommendations, universal vaccination has been currently implemented in 168 countries world wide with an outstanding record of safety and efficacy. The effective implementation of such programmes of vaccination has resulted in a substantial decrease in disease burden, in the carrier rate and in hepatitis B-related morbidity and mortality. A future challenge is to overcome the social and economic hurdles which still hamper the introduction of hepatitis B vaccination on a global scale.

Impact of nucleic acid testing for hepatitis B virus, hepatitis C virus, and human immunodeficiency virus on the safety of blood supply in Italy: a 6-year survey.

BACKGROUND: Nucleic acid testing (NAT) for hepatitis C virus (HCV) and human immunodeficiency virus (HIV) has been implemented in several European countries and in the United States, while hepatitis B virus (HBV) NAT is still being questioned by opinions both in favor and against such an option, depending on the HBV endemicity, health care resources, and expected benefits. STUDY DESIGN AND METHODS: This survey was aimed to assess the NAT impact in improving the safety of blood supply in Italy, 6 years after implementation. The study involved 93 Italian transfusion centers and was carried out in 2001 through 2006. A total of 10,776,288 units were tested for the presence of HCV RNA, 7,932,430 for HIV RNA, and 3,405,497 for HBV DNA, respectively. RESULTS: Twenty-seven donations or 2.5 per million tested were HCV RNA-positive/anti-HCV-negative; 14 or 1.8 per million units tested were HIV RNA-positive/anti-HIV-negative; and 197 or 57.8 per million donations tested were HBV DNA-positive/hepatitis B surface antigen-negative. Of the latter, 8 (2.3/10(6)) were collected from donors in the window phase of infection and 189 (55.5/10(6)) from donors with occult HBV. Sixty-eight percent of the latter donors had hepatitis B surface antibody, 74.5 percent of whom with concentrations considered protective (>or=10 mIU/mL). CONCLUSION: NAT implementation has improved blood safety by reducing the risk of entering 2.5 HCV and 1.8 HIV infectious units per million donations into the blood supply. The yield of NAT in detecting infectious blood before transfusion was higher for HBV than for HCV or HIV. However, the benefit of HBV NAT in terms of avoided HBV-related morbidity and mortality in blood recipients needs to be further evaluated.

Modulation of epitope-specific anti-hepatitis C virus E2 (anti-HCV/E2) antibodies by anti-viral treatment.

The dynamic features of three specific anti-hepatitis C virus (HCV) antibody subpopulations directed against different conformational epitopes of the viral E2 protein (HCV/E2) have been evaluated in patients with primary and persistent HCV infection; the three subpopulations are present in patients infected with different HCV genotypes and have shown a different activity using a pseudovirus neutralization assay (antibodies e301 and e137 exhibiting high neutralizing activity, while antibody e509 enhancement of HCV infectivity). In sequential samples from five patients with primary HCV infection and different virological outcome, all samples tested negative with the single exception of the e509 antibody in a patient not clearing the virus. In sequential samples from 28 patients with persistent infection under treatment with pegylated interferon-alpha plus ribavirin (14 sustained virological responders and 14 non-responders), the therapy did not selectively influence titers of the two neutralizing antibody subpopulations; otherwise, a net increase of the e509 antibody subpopulation related to enhancement of HCV infectivity was observed in non-responders, but not in sustained virological responders (P = 0.0156). This increase was not related to the trend of total anti-HCV/E2 response. The data indicate that a specific antibody response against these epitopes is elicited only late during the infection, thus not influencing virus clearance during primary infection, and that a selective increase of the antibody subpopulation enhancing virus infectivity is observed only in the cohort of patients not responding to antiviral therapy.

Human herpesvirus type 8 infection in an area of Northern Italy with high incidence of classical Kaposi's sarcoma.

Previous studies have reported a large variation in the incidence of classical Kaposi's sarcoma across different Districts of the province of Mantua (Northern Italy). To assess whether such differences might be explained by different anti-HHV8 antibody prevalence, a serological study was conducted in 343 healthy elderly individuals resident in two adjacent Districts, at the highest and the lowest classical Kaposi's sarcoma incidence rate, respectively. Qualitative and quantitative determinations of IgG antibodies against both latent and lytic HHV-8 antigens were performed by indirect immunofluorescence assay. The assay's sensitivity was studied in 26 patients with classical Kaposi's sarcoma. Overall, anti-HHV8 antibodies were detected in 25 out of 26 patients (96%), confirming the high sensitivity of this assay. The prevalence of anti-HHV-8 antibodies was higher among individuals living in the District had a high incidence of classical Kaposi's sarcoma compared to those living in the District with low incidence (19.4% vs 9.8%, and 15.9% vs 8%; P<0.05, for latent and lytic antibodies, respectively). Anti-lytic antibody GMT was higher in people living in the District at high incidence rate compared to those of the other area (328.9 vs. 180.4; P<0.01). A higher prevalence of HHV-8 infection was found among persons living in municipalities surrounded by watercourses (OR 2.2, 95% CI: 1.10-4.32). In conclusion, variation in HHV-8 prevalence appears to explain differences in the incidence rates of classical Kaposi's sarcoma observed in different areas of the province.

Development of a peptide nucleic acid array platform for the detection of genetically modified organisms in food.

Two previously developed platforms, a multiplex polymerase chain reaction (PCR) and a peptide nucleic acid (PNA) array, the former allowing for the simultaneous detection of five transgenes and two endogenous controls in food and feed matrices and the latter for the assessment of the identity of amplified PCR products, were combined in order to develop a PNA array device for the screening of genetically modified organisms (GMOs) in food. PNA probes were opportunely designed, synthesized, and deposited on commercial slides. The length of the probes as well as the distance of the probes from the surface were evaluated and found to be critical points. The most suitable probes were found to be 15-mer PNAs linked to the slide surface by means of two 2-(2-aminoethoxy)ethoxyacetic acids as spacers. The device was tested on a model system constituted by flour samples containing a mixture of standards at known concentrations of transgenic material, in particular Roundup Ready soybean and Bt11, Bt176, Mon810, and GA21 maize: The DNA was amplified using the specific multiplex PCR method and tested on the PNA array. The method proposed was found to be able to correctly identify every GMO present in the tested samples.

Long-term outcome (35 years) of hepatitis C after acquisition of infection through mini transfusions of blood given at birth.

Long-term follow up studies of hepatitis C virus (HCV) infection rarely exceed 20-25 yr. We studied the outcome of HCV infection in 35-yr-old adults infected at birth (1968) through mini transfusions of blood. A retrospective-prospective study was carried out. The cohort included 31 individuals who were given mini blood transfusions (21-30 ml) collected from a donor subsequently revealed to be HCV infected. At enrollment (1998), 18 of 31 (58.1%) recipients had anti-HCV antibody and 16 (88.9%) of them were HCV-RNA positive. All viremic recipients and the infectious donor had the same genotype 1b. Sequence analysis of E1/E2 and NS5b regions, coupled with phylogenetic analysis, indicated that HCV isolates from donor/recipients were linked. Eleven of the 16 viremic recipients gave consent to liver biopsy. Nine had no fibrosis or mild portal fibrosis and 2 had either discrete (Ishak's staging 3) or marked (Ishak's staging 4) fibrosis. During the prospective follow-up period (1998-2003), 2 patients were given therapy, one of whom achieved sustained clinical and virologic response. A second biopsy, performed in 5 patients at a 5 yr interval, revealed no substantial modifications in 4 cases and progression from absence of fibrosis to mild portal fibrosis in the fifth. In conclusion, taking into account the limited study sample, these findings suggest that HCV infection acquired early in life shows a slow progression and mild outcome during the first 35 yr of infection.

Total HCV core antigen assay: a new marker of hepatitis C viremia for monitoring the progress of therapy.

The ability of the total hepatitis C virus (HCV) core antigen assay was evaluated for monitoring the therapeutic responses of HCV-infected patients treated with interferon. The ability to detect and quantitate an independent structural protein component of HCV, in the presence of circulating antibodies, makes this assay a valuable new tool in diagnosis and treatment monitoring. Measurement of total core antigen showed a strong dynamic correlation with HCV RNA data and may serve as an alternative direct marker of viral infection. In addition, with the advent of additional treatment protocols, a rapid, reliable assay for changes in HCV load may permit more frequent patient assessment and tailoring of the therapeutic regimen.

Outcome of an outbreak of acute hepatitis C among healthy volunteers participating in pharmacokinetics studies.

We identified 15 patients with acute hepatitis C (AHC) among 29 healthy volunteers participating in 2 consecutive pharmacokinetics studies. Molecular techniques were used to determine the relatedness of viral strains, whereas clinical and virologic follow-up was started to establish the course and outcome of the acute infection. After presentation, serum liver enzymes and HCV RNA were monitored weekly for 4 months, then monthly for at least 12 months. Liver biopsy was performed 6 to 12 months after AHC diagnosis. Phylogenetic analysis of coding regions for the envelope glycoproteins E1 and E2 was performed. At presentation, all 15 patients tested HCV RNA-positive and had HCV genotype 2c. Phylogenetic analysis indicated a common source of infection. Fourteen patients agreed to be followed prospectively. Infection resolved spontaneously in 8 patients, HCV RNA becoming undetectable by 4 to 5 months after the presumed time of infection in 5 of them and by 8, 13, and 24 months in the remaining 3. Six patients developed chronic infection. Liver biopsies performed in 9 subjects who were HCV RNA-positive 6 months after AHC diagnosis revealed that the prevalent histologic finding was lobular inflammation. In conclusion, our homogeneous cohort showed a wide spectrum of clinical, virologic and histologic features, and, more importantly, short-term outcome differed noticeably despite the common source of infection.

Hepatitis B in Italy: where we are ten years after the introduction of mass vaccination.

In Italy, a program of vaccination against hepatitis B targeted at the immunisation of persons at high risk began in 1983. In 1991, vaccination became mandatory for all newborns and adolescents. Since then, the vaccine has been given to more than 10 million children, with an outstanding record of safety and efficacy. The coverage rate is globally around 94%, with differences between the Northern and Southern regions, with the latter having the lower acceptance rate. According to the National Surveillance System (SEIEVA), the incidence of acute hepatitis B per 10(5) inhabitants declined from 5.4 in 1990 to 2 in 2000. The reduction was even greater among 15-24-year-old individuals, where the incidence rate per 10(5) decreased from 17.3 to 2 in the same period. In parallel with the decline of hepatitis B, hepatitis delta has also declined significantly. Catch-up immunisation of unvaccinated adolescents, as well as an effort to improve the vaccination coverage rate in high-risk groups, are required to ameliorate the efficacy of the vaccination campaign. Routine administration of booster doses of vaccine is not considered necessary to sustain immunity in immunocompetent persons.