The persistence of seroprotective levels of antibodies after vaccination with PreHevbrio, a 3-antigen hepatitis B vaccine.

Prevention of hepatitis B virus (HBV) infection by vaccination can potentially eliminate HBV-related diseases. PreHevbrio™/PreHevbri® is a 3-antigen (S, preS1, preS2) HBV vaccine (3A-HBV) recently licensed for adults in the US, EU and Canada. This study evaluated antibody persistence in a subset of fully vaccinated and seroprotected (anti-HBs ≥ 10 mIU/mL) Finnish participants from the phase 3 trial (PROTECT) of 3A-HBV versus single-antigen HBV vaccine (1A-HBV). 465/528 eligible subjects were enrolled (3A-HBV: 244; 1A-HBV: 221). Baseline characteristics were balanced. After 2.5 years, more 3A-HBV subjects remained seroprotected (88.1 % [95 %CI: 84.1,92.2]) versus 1A-HBV (72.4 % [95 %CI: 66.6,78.3)], p < 0.0001) and had higher mean anti-HBs [1382.9 mIU/mL (95 %CI: 1013.8,1751.9) versus 252.6 mIU/mL (95 %CI: 127.5,377.6), p < 0.0001]. In multiple variable logistic regression analysis including age, vaccine, initial vaccine response, sex and BMI, only higher post dose 3 (Day 196) antibody titers significantly reduced the odds of losing seroprotection.

Immunogenicity and safety of a quadrivalent meningococcal tetanus toxoid-conjugate vaccine (MenACYW-TT) vs. a licensed quadrivalent meningococcal tetanus toxoid-conjugate vaccine in meningococcal vaccine-naïve and meningococcal C conjugate vaccine-primed toddlers: a phase III randomised study.

Vaccination remains the best strategy to reduce invasive meningococcal disease. This study evaluated an investigational tetanus toxoid-conjugate quadrivalent meningococcal vaccine (MenACYW-TT) vs. a licensed tetanus toxoid-conjugate quadrivalent meningococcal vaccine (MCV4-TT) (NCT02955797). Healthy toddlers aged 12-23 months were included if they were either meningococcal vaccine-naïve or MenC conjugate (MCC) vaccine-primed (≥1 dose of MCC prior to 12 months of age). Vaccine-naïve participants were randomised 1:1 to either MenACYW-TT (n = 306) or MCV4-TT (n = 306). MCC-primed participants were randomised 2:1 to MenACYW-TT (n = 203) or MCV4-TT (n = 103). Antibody titres against each of the four meningococcal serogroups were measured by serum bactericidal antibody assay using the human complement. The co-primary objectives of this study were to demonstrate the non-inferiority of MenACYW-TT to MCV4-TT in terms of seroprotection (titres ≥1:8) at Day 30 in both vaccine-naïve and all participants (vaccine-naïve and MCC-primed groups pooled). The immune response for all four serogroups to MenACYW-TT was non-inferior to MCV4-TT in vaccine-naïve participants (seroprotection: range 83.6-99.3% and 81.4-91.6%, respectively) and all participants (seroprotection: range 83.6-99.3% and 81.4-98.0%, respectively). The safety profiles of both vaccines were comparable. MenACYW-TT was well-tolerated and demonstrated non-inferior immunogenicity when administered to MCC vaccine-primed and vaccine-naïve toddlers.

Rotavirus capsid VP6 tubular and spherical nanostructures act as local adjuvants when co-delivered with norovirus VLPs.

A subunit protein vaccine candidate based on norovirus (NoV) virus-like particles (VLPs) and rotavirus (RV) VP6 protein against acute childhood gastroenteritis has been proposed recently. RV VP6 forms different oligomeric nanostructures, including tubes and spheres when expressed in vitro, which are highly immunogenic in different animal models. We have shown recently that recombinant VP6 nanotubes have an adjuvant effect on immunogenicity of NoV VLPs in mice. In this study, we investigated if the adjuvant effect is dependent upon a VP6 dose or different VP6 structural assemblies. In addition, local and systemic adjuvant effects as well as requirements for antigen co-delivery and co-localization were studied. The magnitude and functionality of NoV GII.4-specific antibodies and T cell responses were tested in mice immunized with GII.4 VLPs alone or different combinations of VLPs and VP6. A VP6 dose-dependent adjuvant effect on GII.4-specific antibody responses was observed. The adjuvant effect was found to be strictly dependent upon co-administration of NoV GII.4 VLPs and VP6 at the same anatomic site and at the same time. However, the adjuvant effect was not dependent on the types of oligomers used, as both nanotubes and nanospheres exerted adjuvant effect on GII.4-specific antibody generation and, for the first time, T cell immunity. These findings elucidate the mechanisms of VP6 adjuvant effect in vivo and support its use as an adjuvant in a combination NoV and RV vaccine.

Rotavirus vaccination in Europe: drivers and barriers.

Rotavirus gastroenteritis is a vaccine-preventable disease that confers a high medical and economic burden in more developed countries and can be fatal in less developed countries. Two vaccines with high efficacy and good safety profiles were approved and made available in Europe in 2006. We present an overview of the status of rotavirus vaccination in Europe. We discuss the drivers (including high effectiveness and effect of universal rotavirus vaccination) and barriers (including low awareness of disease burden, perception of unfavourable cost-effectiveness, and potential safety concerns) to the implementation of universal rotavirus vaccination in Europe. By February, 2014, national universal rotavirus vaccination had been implemented in Belgium, Luxembourg, Austria, Finland, Greece, Luxembourg, Norway, and the UK. Four other German states have issued recommendations and reimbursement is provided by sickness funds. Other countries were at various stages of recommending or implementing universal rotavirus vaccination.

Characterization and immunogenicity of norovirus capsid-derived virus-like particles purified by anion exchange chromatography.

Recombinant baculovirus (BV) expression systems are widely applied in the production of viral capsid proteins and virus-like particles (VLPs) for use as immunogens and vaccine candidates. Traditional density gradient purification of VLPs does not enable complete elimination of BV-derived impurities, including live viruses, envelope glycoprotein gp64 and baculoviral DNA. We used an additional purification system based on ionic strength to purify norovirus (NoV) GII-4 capsid-derived VLPs. The anion exchange chromatography purification led to highly purified VLPs free from BV impurities with intact morphology. In addition, highly purified VLPs induced strong NoV-specific antibody responses in BALB/c mice. Here, we describe a method for NoV VLP purification and several methods for determining their purity, including quantitative PCR for BV DNA detection.

Rotavirus vaccination: a concise review.

Live attenuated oral rotavirus vaccines were tested for proof-of-concept in the early 1980s, the first vaccine (RotaShield, Wyeth) was introduced in 1998 but was subsequently withdrawn because of association with intussusception, and the two currently licensed vaccine (Rotarix, GlaxoSmithKline, and RotaTeq, Merck) were introduced in 2006. Before licensure both vaccines were extensively tested for safety (for intussusception) and efficacy in trials comprising in over 60,000 infants each. Rotarix is a single-strain human rotavirus vaccine (RV1) and RotaTeq is a combination of five bovine-human reassortant rotaviruses (RV5). Although the composition of the two vaccines is different, their field effectiveness and, largely, mechanism of action are similar. Both prevent effectively severe rotavirus gastroenteritis (RVGE) but are less efficacious against mild RVGE or rotavirus infection. Field effectiveness of these vaccines in Europe and the USA against severe RVGE has been above 90% and in Latin America around 80%. Trials in Africa have yielded efficacy rates between 50 and 80%. Rotavirus vaccination has been introduced into the national immunization programmes of about 20 countries in Latin America, with Brazil and Mexico as leading countries, as well as in the USA, Australia and South Africa. Introduction into other African countries will start in 2012. In Europe, Belgium, Luxembourg, Austria and Finland and five federal states of Germany have introduced universal rotavirus vaccination. The reasons for the slow progress in Europe include low mortality from RVGE, unfavourable cost-benefit calculations in some countries, and concerns that still exist over intussusception.

Norovirus genotypes in endemic acute gastroenteritis of infants and children in Finland between 1994 and 2007.

Noroviruses are, after rotaviruses, the second most common causative agents of acute gastroenteritis in young children. We studied norovirus genotypes in faecal specimens collected from Finnish children followed-up prospectively in rotavirus vaccine trials. Almost 5000 faecal specimens collected from cases of acute gastroenteritis were examined using reverse transcriptase-PCR. A total of 1172 cases (25% of all acute gastroenteritis) were associated with noroviruses. Of these, 96% were genogroup GII. GII.4 was the most common genotype (46%) throughout the study period but the proportion of this genotype varied in different norovirus epidemic seasons. Additional norovirus genotypes detected were: GII.7 (15%), GII.3 (14%), GII.1 (9%), GII.b (7%), GII.2 (3%), and GI.3 (2%). GII.4 dominated during the following years: 1998-1999 (75%), 2002-2003 (88%) and 2006-2007 (98%) while recombinant genotype GII.b was dominant between 2003 and 2004 (83%). In conclusion, genotypes GII.4 and GIIb have emerged as predominant norovirus genotypes in endemic gastroenteritis affecting young infants and children in Finland.

Prevalence of norovirus GII-4 antibodies in Finnish children.

Noroviruses (NoVs) are the second most common cause of viral gastroenteritis after rotavirus in children. NoV genotype GII-4 has emerged as the major type not only in outbreaks of NoV gastroenteritis but also endemic gastroenteritis among infants and young children worldwide. Using baculovirus-insect cell system virus-like particles (VLPs) of NoV genotype GII-4 and an uncommon genotype GII-12 were produced. These VLPs were used in enzyme-linked immunosorbent assays (ELISA) for detection of NoV-specific immunoglobulin G (IgG) and IgA antibodies in 492 serum specimens from Finnish children 0-14 years of age collected between 2006 and 2008. NoV IgG antibody prevalence was 47.3% in the age group 7-23 months and increased up to 91.2% after the age of 5 years. Avidity of NoV IgG antibodies was low in the primary infections while high avidity antibodies were detected in the recurrent infections of the older children. In GII-4 infections, the homologous antibody response to GII-4 VLPs was stronger than to GII-12 VLPs but cross-reactivity between GII-4 and GII-12 was observed. Binding of GII-4 VLPs to a putative carbohydrate antigen receptor H-type 3 could be blocked by sera from children not infected with NoV during a waterborne outbreak of acute gastroenteritis. Therefore, protection against NoV infection correlated with strong blocking activity.

Immunogenicity, reactogenicity and safety of the human rotavirus vaccine RIX4414 oral suspension (liquid formulation) in Finnish infants.

The lyophilized formulation of a human rotavirus vaccine, Rotarix™ (RIX4414) is highly immunogenic. In order to comply with the World Health Organization's (WHO) recommendation, a liquid formulation of the vaccine that does not require reconstitution was developed. The immunogenicity, reactogenicity and safety of the liquid formulation were compared with lyophilized formulation in two Finnish studies. In Study A infants aged 6-12 weeks received two doses of the lyophilized or liquid formulation of the vaccine or placebo following a 0,1 month schedule. In Study B, infants aged 10-17 weeks received two doses of either liquid or lyophilized formulation of the vaccine. In both studies, anti-rotavirus IgA antibodies were assessed pre-vaccination and one month post-Dose 2. In Study A, the anti-rotavirus seroconversion rate was 90% (95% CI: 81.2-95.6%) and 83.7% (95% CI: 74.2-90.8%) in the groups that received the liquid and the lyophilized formulation of RIX4414, respectively; the respective anti-rotavirus IgA seroconversion rates in Study B were 88.6% (95% CI: 86.1-90.8%) and 90.5% (95% CI: 86.2-93.8%). Reactogenicity and safety profiles of the two vaccine formulations were similar. Liquid formulation of the rotavirus vaccine allows greater flexibility in supply and reduces logistical costs.

A comparison of methods for purification and concentration of norovirus GII-4 capsid virus-like particles.

Noroviruses (NoVs) are one of the leading causes of acute gastroenteritis worldwide. NoV GII-4 VP1 protein was expressed in a recombinant baculovirus system using Sf9 insect cells. Several methods for purification and concentration of virus-like particles (VLPs) were evaluated. Electron microscopy (EM) and histo-blood group antigen (HBGA) binding assays showed that repeated sucrose gradient purification followed by ultrafiltration resulted in intact VLPs with excellent binding to H type 3 antigens. VLPs were stable for at least 12 months at 4°C, and up to 7 days at ambient temperature. These findings indicate that this method yielded stable and high-quality VLPs.

Mixed viral infections causing acute gastroenteritis in children in a waterborne outbreak.

We examined stool specimens for viral pathogens from 50 children referred to hospital due to acute gastroenteritis (AGE) resulting from consuming drinking water contaminated with sewage in a Finnish community using PCR methods. Rotavirus was detected in 33 (66%), human calicivirus in 31 (62%), and both in 40% of cases. Of the caliciviruses, 20/31 (65%) were noroviruses and 11 (35%) sapoviruses. Furthermore, Aichi virus was detected in 25 (50%), adenovirus in six (12%) and bocavirus in four (8%) cases. Campylobacter jejuni was present in 20 (61%) and Salmonella in four (12%) of the 33 stools cultured for bacteria. On a 20-point scale median severity score of AGE in the 28 hospitalized children was 17; the severity was similar regardless of viruses detected. Bloody diarrhoea occurred only when C. jejuni was present. To conclude, massive exposure to several AGE viruses caused mixed infections and severe AGE regardless of the aetiological agents.

Aichi virus infection in children with acute gastroenteritis in Finland.

Aichi virus has been proposed as a novel causative agent of acute gastroenteritis. In addition to several Asian countries, South America and Africa, Aichi virus has also recently been found in Europe. Our objective was to study the causative role of Aichi virus in children with acute gastroenteritis in Finland. We analysed 595 stool specimens from infants in an efficacy trial of rotavirus vaccine and 468 stool specimens from children in a hospital-based epidemiological and aetiological study of acute gastroenteritis. The screening was done by nested reverse transcription-polymerase chain reaction amplifying a 519-bp segment and a 223-bp segment in the 3CD junction region of non-structural proteins. Aichi virus was detected in five stool samples (0.5%), of which four were co-infections with other gastroenteritis viruses. Two Aichi virus genotypes, A and B, were found. Aichi virus appears to be rare in children with acute gastroenteritis in Finland.

MF59-adjuvanted influenza vaccine (FLUAD) in children: safety and immunogenicity following a second year seasonal vaccination.

After priming with two intramuscular doses of MF59-adjuvanted (Sub/MF59) or split influenza vaccines during the 2006/07 season, 89 healthy children received a third booster dose of the respective vaccine (2007/08 Northern Hemisphere formulation) approximately 1 year later, and were followed up for 6 months post-third injection. Immunogenicity was evaluated on 81 of them by a hemagglutination inhibition (HI) assay before and 3 weeks after vaccination. The Sub/MF59 influenza vaccine was safe and well tolerated following the booster vaccination. Pre-booster HI antibody titers were consistently higher in the Sub/MF59 group than in the comparator group, confirming significantly longer persistence of antibodies after priming with Sub/MF59 vaccine. Post-booster immune responses were significantly higher in the Sub/MF59 group compared with the split group, especially vs. the influenza B strain, which is epidemiologically relevant in the pediatric population. Altogether, these data further support the potential use of MF59-adjuvanted influenza vaccine as a safe and highly immunogenic influenza vaccine for young children.

One-step quantitative RT-PCR for the detection of rotavirus in acute gastroenteritis.

The standard diagnosis of rotavirus gastroenteritis is based on the demonstration of rotavirus antigen in stools using an enzyme immunoassay (EIA). In this study, a one-step quantitative RT-PCR (Q-PCR) was used for sensitive detection of rotavirus in diarrheal stools. The primers and TaqMan probe for the Q-PCR were selected from a highly conserved region of the non-structural protein 3 (NSP3) of rotavirus. After validation, the test was applied to study rotavirus EIA positive (N=25) and EIA negative (N=143) stool specimens from cases of acute gastroenteritis of all degrees of severity in a prospective follow-up cohort of infants from 2 months to 2 years of age. Q-PCR detected all 25 EIA positive rotavirus antigens and seven additional cases that were rotavirus EIA negative, i.e. 28% more rotavirus positive cases than identified by EIA. It is concluded that Q-PCR using primers targeted at NSP3 is a rapid and sensitive method for diagnosing acute rotavirus gastroenteritis.

Efficacy of human rotavirus vaccine against rotavirus gastroenteritis during the first 2 years of life in European infants: randomised, double-blind controlled study.

BACKGROUND: We aimed to assess the efficacy of the oral live attenuated human rotavirus vaccine Rotarix (RIX4414) for prevention of rotavirus gastroenteritis in European infants during their first 2 years of life. METHODS: 3994 study participants were enrolled from six countries and were randomly assigned two oral doses of either RIX4414 (n=2646) or placebo (n=1348), which were coadministered with the first two doses of specific childhood vaccinations. Follow-up for gastroenteritis episodes was undertaken from 2 weeks post-dose two through the two consecutive rotavirus seasons following vaccinations (combined efficacy follow-up period; mean duration 17 months [SD 1.6]). Our primary endpoint was vaccine efficacy against rotavirus gastroenteritis of any severity during the first efficacy follow-up period (2 weeks post-dose two to the end of the first rotavirus season). Stool specimens obtained during gastroenteritis episodes were tested for rotavirus by ELISA and typed by RT-PCR. Episodes scoring 11 or greater on the 20-point Vesikari scale were classified as severe. Analysis was according to protocol. This study is registered with ClinicalTrials.gov, number NCT00140686 (eTrack102247). FINDINGS: 120 infants were excluded from the according-to-protocol analysis. During the first efficacy follow-up period (mean duration 5.7 months [SD 1.2]), 24 of 2572 infants allocated RIX4414 versus 94 of 1302 given placebo had rotavirus gastroenteritis episodes of any severity, resulting in a vaccine efficacy of 87.1% (95% CI 79.6-92.1; p<0.0001). For the combined efficacy follow-up period, vaccine efficacy against severe rotavirus gastroenteritis was 90.4% (85.1-94.1; p<0.0001), for admission owing to rotavirus gastroenteritis 96.0% (83.8-99.5; p<0.0001), and for rotavirus-related medical attention 83.8% (76.8-88.9; p<0.0001), and significant protection against severe rotavirus gastroenteritis by circulating G1, G2, G3, G4, and G9 rotavirus types was shown. INTERPRETATION: In a European setting, two doses of RIX4414 coadministered with childhood vaccines provided high protection against any and severe rotavirus gastroenteritis, with an overall reduction of admissions for gastroenteritis over two consecutive rotavirus epidemic seasons.

Relationship between the incidence of type 1 diabetes and maternal enterovirus antibodies: time trends and geographical variation.

AIMS/HYPOTHESIS: We have previously observed an inverse correlation between the incidence of type 1 diabetes and enterovirus infections in the background population. The aim of this study was to analyse whether maternal enterovirus antibody status, which reflects both the frequency of enterovirus infections and the protection conferred by the mother on the offspring, also correlates with the incidence of type 1 diabetes. METHODS: Maternal enterovirus antibodies were analysed from serum samples taken from pregnant women between 1983 and 2001 in Finland and Sweden using enzyme immunoassay and neutralisation assays. Comparable samples were also taken between 1999 and 2001 in countries with a lower incidence of diabetes (Estonia, Germany, Hungary, Israel, Lithuania, Russia). RESULTS: A clear decrease was observed in maternal enterovirus antibody levels over the past 20 years (p<0.0001). The frequency of enterovirus antibodies was higher in countries with a low or intermediate incidence of type 1 diabetes compared with high-incidence countries (p<0.0001). CONCLUSIONS/INTERPRETATION: These findings are in line with our previous observations supporting the hypothesis that a low frequency of enterovirus infection in the background population increases the susceptibility of young children to the diabetogenic effect of enteroviruses.

Safety and immunogenicity of RIX4414 live attenuated human rotavirus vaccine in adults, toddlers and previously uninfected infants.

A live attenuated human rotavirus (HRV) vaccine, strain RIX4414, was tested sequentially in adults, previously infected toddlers, and previously uninfected infants. A single dose was given to adults and toddlers and found well tolerated. Next, a dose ranging (three different viral concentrations) safety and immunogenicity study was conducted in rotavirus IgA antibody negative infants (N= 192), who received two doses of RIX4414 vaccine or placebo at 2 and 4 months of age. No side effects were seen after vaccination. Specifically, administration of RIX4414 vaccine was not temporally associated with fever, diarrhea, or increase in liver transaminases. Rotavirus IgA seroconversion ranged from 50 to 88% after one dose and from 73 to 96% after two doses, depending on vaccine titer. After the first dose, on days 7-9 post vaccination, between 38 and 60% of the infants shed the vaccine virus, whereas after the second dose only 0 to 13% of the vaccinees shed the vaccine virus. It is concluded that RIX4414 strain HRV vaccine is virtually non-reactogenic and, at high titer, highly immunogenic in susceptible infants.

Screening of rotavirus and adenovirus infections during prolonged hospitalization in a neonatal unit.

UNLABELLED: Rotavirus and adenovirus infections in 308 infants hospitalized for longer than 1 wk, and cases with necrotizing enterocolitis, were screened in a neonatal unit during a 15 mo period, covering two rotavirus epidemics in the community. Altogether, 1020 stool samples were collected weekly until hospital discharge, and in necrotizing enterocolitis cases at the onset of symptoms, and tested for rotavirus and adenovirus by means of enzyme-linked immunosorbent assay. The positive samples were further analysed by polymerase chain reaction. Enzyme-linked immunosorbent assay revealed five adenovirus-positive cases, which were tested negative by polymerase chain reaction. Out of 16 necrotizing enterocolitis cases, one was adenovirus- and another rotavirus positive when tested by polymerase chain reaction, the latter having a concomitant Candida albicans septicaemia. CONCLUSION: Routine rotavirus and adenovirus screening in hospitalized neonates seems to be unnecessary. Viral diagnostic examinations should be considered in patients with necrotizing enterocolitis.