Health and Economic Outcomes of Introducing the New MenB Vaccine (Bexsero) into the Italian Routine Infant Immunisation Programme

Introduction

In January 2013 a novel type of multicomponent protein-based vaccine against group B meningococcal disease was licensed by the European Medicines Agency. With the widespread use of the meningococcal serogroup C conjugate vaccines, serogroup B remains now the major cause of bacterial meningitis and septicaemia in young children in Europe. The aim of this study is to investigate the health and the economic outcomes of MenB vaccine introduction into the Italian routine mass vaccination programme.

Methods

The present work is structured in two main parts. Firstly, we assess the epidemiological burden of group B meningococcal disease using official hospitalisation and notification data from two of the most populated Italian regions (Lombardia and Piemonte) during a 6-year study period (2007-2012). Secondly, we evaluate the cost-effectiveness of the immunisation programme in Italy from the public health payer perspective under base case parameters assumptions and performing a comprehensive sensitivity analysis to assess the robustness and the uncertainty of our model results.

Results

MenB serotype is responsible for 59% of the 341 cases of Invasive Meningococcal Disease in Lombardia and Piemonte. Incidence rate for MenB infection is estimated to be 0.21/100,000/y resulting at the highest level in children ≤4 years of age. Although the new MenB vaccine can potentially prevent about one third of the disease cases in the Italian population, model results show this strategy is unlikely to be cost-effective (ICER value over €350,000/QALY) with a vaccine that prevents disease only. These results are robust under most of the sensitivity scenarios except when allowing for lower discount rates.

Discussion

The introduction of the novel vaccine into the routine immunisation schedule needs to be carefully evaluated. The new MenB vaccine has the potential to reduce the disease burden at the population level. However, from the Italian Health Service perspective, the immunisation programme is unlikely to be cost-effective at the current incidence levels and vaccine price.     

Live Cell Analysis and Mathematical Modeling Identify Determinants of Attenuation of Dengue Virus 2’-O-Methylation Mutant

Dengue virus (DENV) is the most common mosquito-transmitted virus infecting ~390 million people worldwide. In spite of this high medical relevance, neither a vaccine nor antiviral therapy is currently available. DENV elicits a strong interferon (IFN) response in infected cells, but at the same time actively counteracts IFN production and signaling. Although the kinetics of activation of this innate antiviral defense and the timing of viral counteraction critically determine the magnitude of infection and thus disease, quantitative and kinetic analyses are lacking and it remains poorly understood how DENV spreads in IFN-competent cell systems. To dissect the dynamics of replication versus antiviral defense at the single cell level, we generated a fully viable reporter DENV and host cells with authentic reporters for IFN-stimulated antiviral genes. We find that IFN controls DENV infection in a kinetically determined manner that at the single cell level is highly heterogeneous and stochastic. Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN. By contrast, a vaccine candidate DENV mutant, which lacks 2’-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. Through mathematical modeling of time-resolved data and validation experiments we show that the primary determinant for attenuation is the accelerated kinetics of IFN production. This rapid induction triggered by mutant DENV precedes establishment of IFN-resistance in infected cells, thus causing a massive reduction of virus production rate. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. In conclusion, these results show that attenuation of the 2’-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.     

Microevolution of Anthrax from a Young Ancestor (M.A.Y.A.) Suggests a Soil-Borne Life Cycle of Bacillus anthracis

During an anthrax outbreak at the Pollino National Park (Basilicata, Italy) in 2004, diseased cattle were buried and from these anthrax-foci Bacillus anthracis endospores still diffuse to the surface resulting in local accumulations. Recent data suggest that B. anthracis multiplies in soil outside the animal-host body. This notion is supported by the frequent isolation of B. anthracis from soil lacking one or both virulence plasmids. Such strains represent an evolutionary dead end, as they are likely no longer able to successfully infect new hosts. This loss of virulence plasmids is explained most simply by postulating a soil-borne life cycle of the pathogen. To test this hypothesis we investigated possible microevolution at two natural anthrax foci from the 2004 outbreak. If valid, then genotypes of strains isolated from near the surface at these foci should be on a different evolutionary trajectory from those below residing in deeper-laying horizons close to the carcass. Thus, the genetic diversity of B. anthracis isolates was compared conducting Progressive Hierarchical Resolving Assays using Nucleic Acids (PHRANA) and next generation Whole Genome Sequencing (WGS). PHRANA was not discriminatory enough to resolve the fine genetic relationships between the isolates. Conversely, WGS of nine isolates from near-surface and nine from near-carcass revealed five isolate specific SNPs, four of which were found only in different near-surface isolates. In support of our hypothesis, one surface-isolate lacked plasmid pXO1 and also harbored one of the unique SNPs. Taken together, our results suggest a limited soil-borne life cycle of B. anthracis.     

Genetic Origins of Lactase Persistence and the Spread of Pastoralism in Africa

In humans, the ability to digest lactose, the sugar in milk, declines after weaning because of decreasing levels of the enzyme lactase-phlorizin hydrolase, encoded by LCT. However, some individuals maintain high enzyme amounts and are able to digest lactose into adulthood (i.e., they have the lactase-persistence [LP] trait). It is thought that selection has played a major role in maintaining this genetically determined phenotypic trait in different human populations that practice pastoralism. To identify variants associated with the LP trait and to study its evolutionary history in Africa, we sequenced MCM6 introns 9 and 13 and ∼2 kb of the LCT promoter region in 819 individuals from 63 African populations and in 154 non-Africans from nine populations. We also genotyped four microsatellites in an ∼198 kb region in a subset of 252 individuals to reconstruct the origin and spread of LP-associated variants in Africa. Additionally, we examined the association between LP and genetic variability at candidate regulatory regions in 513 individuals from eastern Africa. Our analyses confirmed the association between the LP trait and three common variants in intron 13 (C-14010, G-13907, and G-13915). Furthermore, we identified two additional LP-associated SNPs in intron 13 and the promoter region (G-12962 and T-956, respectively). Using neutrality tests based on the allele frequency spectrum and long-range linkage disequilibrium, we detected strong signatures of recent positive selection in eastern African populations and the Fulani from central Africa. In addition, haplotype analysis supported an eastern African origin of the C-14010 LP-associated mutation in southern Africa.     

SAR study and conformational analysis of a series of novel peptide G protein‐coupled receptor kinase 2 inhibitors

G protein‐coupled receptor kinase 2 (GRK2) plays a central role in the cellular transduction network. In particular, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. Thereby, its inhibition offers a potential therapeutic solution to several pathological conditions. In the present study, we performed a SAR study and a NMR conformational analysis of peptides derived from HJ loop of GRK2 and able to selectively inhibit GRK2. From Ala‐scan and d ‐Ala point replacement, we found that Arg residues don't affect the inhibitory properties, while a d ‐amino acid at position 5 is key to the activity. Conformational analysis identified two β‐turns that involve N‐terminal residues, followed by a short extended region. These information can help the design of peptides and peptido‐mimetics with enhanced GRK2 inhibition properties. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 121–128, 2014.     

Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders

The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N‐ethylmaleimide‐sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol‐enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders.     

Investigating the candidacy of LPS-based glycoconjugates to prevent invasive meningococcal disease: immunology of glycoconjugates with high carbohydrate loading

We investigated the immune responses of rabbits that were immunised with lipopolysaccharide (LPS)-based glycoconjugates by measuring the reactivity of the derived sera to a panel of selected wild-type and mutant strains of Neisseria meningitidis. In all cases, high titers of antibodies capable of recognising LPS elaborating the identical structure as presented on the immunising glycoconjugate were obtained, and in most cases the derived sera also recognised heterologous strains including wild-type, but at lower titers. However, although serum bactericidal antibodies were consistently obtained against strains elaborating the same LPS structure as the immunising antigen, this functional response was not observed against wild-type strains. We identified several potentially competing neo-epitopes that had been introduced via our conjugation strategies, which might compete with the conserved inner core oligosaccharide target region, thus reducing the antibody titers to epitopes which could facilitate bactericidal killing. This study has therefore identified key factors that are crucial to control in order to increase the likelihood of obtaining bactericidal antibodies to wild-type meningococcal cells with LPS-derived glycoconjugates. Glycoconjugates utilised in this study, have been found to contain epitopes that do not contribute to the derivation of antibodies that may facilitate bactericidal killing of wild-type strains and must be avoided in future LPS-based glycoconjugate preparations.     

Autophagy is defective in collagen VI muscular dystrophies, and its reactivation rescues myofiber degeneration

Autophagy is crucial in the turnover of cell components, and clearance of damaged organelles by the autophagic-lysosomal pathway is essential for tissue homeostasis. Defects of this degradative system have a role in various diseases, but little is known about autophagy in muscular dystrophies. We have previously found that muscular dystrophies linked to collagen VI deficiency show dysfunctional mitochondria and spontaneous apoptosis, leading to myofiber degeneration. Here we demonstrate that this persistence of abnormal organelles and apoptosis are caused by defective autophagy. Skeletal muscles of collagen VI-knockout (Col6a1(-/-)) mice had impaired autophagic flux, which matched the lower induction of beclin-1 and BCL-2/adenovirus E1B-interacting protein-3 (Bnip3) and the lack of autophagosomes after starvation. Forced activation of autophagy by genetic, dietary and pharmacological approaches restored myofiber survival and ameliorated the dystrophic phenotype of Col6a1(-/-) mice. Furthermore, muscle biopsies from subjects with Bethlem myopathy or Ullrich congenital muscular dystrophy had reduced protein amounts of beclin-1 and Bnip3. These findings indicate that defective activation of the autophagic machinery is pathogenic in some congenital muscular dystrophies.     

Quantitative HIV-1 proviral DNA detection: a multicentre analysis

Despite the widespread use of molecular biology techniques, standardized methods for the measurement of HIV-1 proviral DNA are currently lacking and several discordant results are still present in different studies. To assess the clinical meaning of the proviral DNA load, a study group comprising seven different laboratories was set up to standardize a HIV-1 proviral DNA quantification method able to assess the DNA proviral load of the most relevant circulating HIV-1 subtypes. Reference samples (24 cellular samples infected with HIV-1 clade B, and 40 samples of peripheral blood mononuclear cells containing different concentrations of plasmids expressing different HIV-1 clades) were distributed and tested blindly. All laboratories employed hTERT gene as housekeeping gene and primers within the gag gene to quantify different HIV-1 clades. Inter-laboratory results did not differ statistically but showed only minor variations concerning HIV-1 DNA amounts and different HIV clades, with a good agreement among the laboratories participating in the study. Since test standardization represents a key step for future application in clinical practice, further studies of the patients' samples are in progress to establish the real meaning and utility of the proviral DNA load for clinical management of HIV-1 infected patients.