Development of a Virosomal RSV Vaccine Containing 3D-PHAD® Adjuvant: Formulation, Composition, and Long-Term Stability.

PURPOSE: Characterization of virosomes, in late stage preclinical development as vaccines for Respiratory Syncytial Virus (RSV), with a membrane-incorporated synthetic monophosphoryl lipid A, 3D-PHAD® adjuvant. METHODS: Virosomes were initially formed by contacting a lipid film containing 3D-PHAD® with viral membranes solubilized with the short chain phospholipid DCPC, followed by dialysis, later by adding solubilized 3D-PHAD to viral membranes, or to preformed virosomes from DMSO. RESULTS: Virosomes formed from lipid films contained the membrane glycoproteins G and F, at similar F to G ratios but lower concentrations than in virus, and the added lipids, but only a fraction of the 3D-PHAD®. By single particle tracking (SPT), the virosome size distribution resembled that seen by cryo-electron microscopy, but dynamic light scattering showed much larger particles. These differences were caused by small virosome aggregates. Measured by SPT, virosomes were stable for 300 days. 3DPHAD ® incorporation in virosomes could be enhanced by providing the adjuvant from DCPC solubilized stock, but also by adding DMSO dissolved adjuvant to pre-formed virosomes. Virosomes with 0.1 mg/mg of 3D-PHAD®/viral protein from DMSO induced antibody titers similar to those by virosomes containing 0.2 mg/mg of DCPC-solubilized 3D-PHAD®. CONCLUSIONS: Stable 3D-PHAD® adjuvanted RSV virosomes can be formulated.

Influenza vaccination in healthcare workers; comparison of side effects and preferred route of administration of intradermal versus intramuscular administration.

OBJECTIVE: To explore the nature and severity of side effects and future preference of intradermal versus intramuscular influenza vaccination in healthcare workers. DESIGN: Prospective cohort study. SETTING: Two University Medical Centers in The Netherlands. PARTICIPANTS: Healthcare workers receiving an influenza vaccination. METHODS: Healthcare workers that were vaccinated during the influenza vaccination season of 2012-2013 were approached for participation in a questionnaire study. The questionnaire was divided into two parts. The first part had to be answered directly after vaccination and the second part two weeks after vaccination. The motivation for vaccine uptake, whether or not the HCWs had direct contact with patients and the prevalence and severity of local and systemic side effects of influenza vaccination were explored. In addition, it was assessed how participants experienced the vaccination and which type of administration they preferred for future vaccination. RESULTS: Side effects of vaccination were more prevalent in the intradermal group versus the intramuscular group (56% versus 26%, p<0.001). Local side effects were perceived as more severe in healthcare workers receiving the intradermal vaccine. Directly after vaccination, healthcare workers preferred the intradermal vaccination. Two weeks after vaccination both types of vaccine were equally appreciated. CONCLUSIONS: This study shows that there are significant differences in the nature and severity of side effects upon intramuscular and intradermal influenza vaccination. This difference did not result in a preference among the vaccinated subjects for one type of vaccine.

Cost-effectiveness of the prophylactic HPV vaccine: an application to the Netherlands taking non-cervical cancers and cross-protection into account.

Despite an effective screening programme, 600-700 women are still diagnosed with cervical cancer in the Netherlands each year. In 2009 a prophylactic vaccine against HPV-type 16 and 18 was implemented in the national immunisation programme to decrease the incidence of cervical cancer. There is evidence that infections with several oncogenic HPV types other than the vaccine types 16 and 18 are also prevented by vaccination, also known as cross-protection. Besides cervical cancer, HPV can also cause cancers at other sites such as the oropharynx, vulva, vagina and the anus/anal area. In this study we estimated the maximum health and economic benefits of vaccinating 12-year old girls against infection with HPV, taking cross-protection and non-cervical cancers into account. In the base-case, we found an incremental cost ratio (ICER) of €5815 per quality adjusted life year (QALY). Robustness of this result was examined in sensitivity analysis. The ICER proved to be most sensitive to vaccine price, discounting rates, costs of cervical cancer and to variation in the disutility of cervical cancer.

Cost-effectiveness of potential infant vaccination against respiratory syncytial virus infection in The Netherlands.

INTRODUCTION: Respiratory syncytial virus (RSV) infection is one of the major causes of respiratory illness in infants, infecting virtually every child before the age of 2 years. Currently, several Phase 1 trials with RSV vaccines in infants are ongoing or have been completed. As yet, no efficacy estimates are available for these vaccine candidates. Nevertheless, cost-effectiveness estimates might be informative to enable preliminary positioning of an RSV vaccine. METHODS: A decision analysis model was developed in which a Dutch birth cohort was followed for 12 months. A number of potential vaccination strategies were reviewed such as vaccination at specific ages, a two- or three-dosing scheme and seasonal vaccination versus year-round vaccination. The impact of the assumptions made was explored in various sensitivity analyses, including probabilistic analysis. Outcome measures included the number of GP visits, hospitalizations and deaths, costs, quality-adjusted life years and incremental cost-effectiveness ratios (ICERs). RESULTS: Currently, without vaccination, an annual number of 28,738 of RSV-related GP visits, 1623 hospitalizations, and 4.5 deaths are estimated in children in the age of 0-1 year. The total annual cost to society of RSV in the non-vaccination scenario is €7.7 million (95%CI: 1.7-16.7) and the annual disease burden is estimated at 597 QALYs (95%CI: 133-1319). In case all infants would be offered a potentially safe and effective 3-dose RSV vaccination scheme at the age of 0, 1 and 3 months, the total annual net costs were estimated to increase to €21.2 million, but 544 hospitalizations and 1.5 deaths would be averted. The ICER was estimated at €34,142 (95%CI: € 21,652-€ 87,766) per QALY gained. A reduced dose schedule, seasonal vaccination, and consideration of out-of-pocket expenses all resulted in more favorable ICER values, whereas a reduced vaccine efficacy or a delay in the timing of vaccination resulted in less favorable ICERs. DISCUSSION: Our model used recently updated estimates on the burden of RSV disease in children and it included plausible utilities. However, due to the absence of clinical trial data, a number of crucial assumptions had to be made related to the characteristics of potential RSV vaccine. The outcomes of our modeling exercise show that vaccination of infants against RSV might be cost-effective. However, clinical trial data are warranted.

Cost-effectiveness of prophylactic vaccination against human papillomavirus 16/18 for the prevention of cervical cancer: adaptation of an existing cohort model to the situation in the Netherlands.

Cervical cancer is one of the most prevalent cancers among women worldwide. Implementation of an HPV-vaccination strategy targeting the major oncogenic types 16 and 18 that cause cervical cancer is generally expected to significantly reduce the burden of cervical cancer disease. Here we estimate the costs, savings and health gains with the addition of HPV-16/18 vaccination to the already existing Dutch screening programme. In the base-case analysis, it was estimated that implementation of an HPV-16/18 vaccine would result in an incremental cost-effectiveness ratio (ICER) of euro22,700 per life-year gained (LYG). In sensitivity analysis, the robustness of our finding of favourable cost-effectiveness was established. The ICER appeared sensitive to the vaccine price, discount rate and duration of vaccine-induced protection. From our results, it validly follows that immunization of 12-year-old Dutch girls against HPV-16/18 infection is a cost-effective strategy for protecting against cervical cancer.

Wegener's granulomatosis patients show an adequate antibody response to influenza vaccination.

OBJECTIVES: Wegener's granulomatosis (WG) is a systemic vasculitis characterised by relapsing and remitting disease activity. Immunosuppressive drugs are used to control disease, but increase susceptibility to infection. Therefore, influenza vaccination should be considered in WG patients. This study was performed to assess the immunogenicity of influenza vaccination in WG patients. METHODS: A randomised, controlled trial was performed in WG patients with quiescent disease, defined as a Birmingham vasculitis activity score (BVAS) less than 2. Patients were randomly assigned to receive influenza vaccination (n = 49) or to participate as controls (n = 23). In addition, healthy controls (n = 49) were vaccinated. At entry and at 1 and 3-4 months after entry, antibody responses to vaccination were determined. Furthermore, disease activity was measured (BVAS), adverse effects were recorded and antineutrophil cytoplasmic autoantibody (ANCA) titres were determined. RESULTS: WG patients achieved high seroprotection rates to all three influenza strains, comparable with healthy controls. Only the A/H1N1 strain patients had a lower seroconversion rate (p = 0.002) and geometric mean titre (p = 0.037) than controls. After 1 month, one control and one vaccinated WG patient had developed active disease. At 3-4 months, two additional control patients had developed active disease compared with none of the vaccinated patients (p = 0.099). Vaccination did not influence ANCA titres. Adverse effects did not differ between patients and healthy controls. CONCLUSIONS: Influenza vaccination in WG patients with quiescent disease induced a sufficient antibody response. TRIAL REGISTRATION NUMBER: NTR1130.

Excess drug prescriptions during influenza and RSV seasons in the Netherlands: potential implications for extended influenza vaccination.

Influenza and respiratory syncytial virus (RSV) infections are responsible for considerable morbidity, mortality and health-care resource use. For the Netherlands, we estimated age and risk-group specific numbers of antibiotics, otologicals and cardiovascular prescriptions per 10,000 person-years during periods with elevated activity of influenza or RSV, and compared these with peri-season rates. Data were taken from the University of Groningen in-house prescription database (www.iadb.nl) and virological surveillance for the period 1998-2006. During influenza and RSV periods excess antibiotic prescriptions were estimated for all age groups. In the age groups 0-1 and 2-4 years, excess antibiotic prescriptions during periods with elevated RSV activity (65% and 59% of peri-seasonal rates) exceeded the surpluses estimated during the influenza-activity periods (24% and 34% of peri-seasonal rates) while for otologicals excess prescriptions were higher for influenza (22% and 27%) than for RSV (14% and 17%). Among persons of 50 years and older, notably those without medical high-risk conditions, excess prescriptions for cardiovascular medications were estimated during the influenza periods at approximately 10% (this was also already seen in persons aged 45-49). Our results may have implications for influenza vaccination policies. In particular, extension of influenza vaccination to groups of non-elderly adults and young children may lower excess prescriptions during these influenza periods for all three types of drug prescriptions investigated.

The use of health economics to guide drug development decisions: Determining optimal values for an RSV-vaccine in a model-based scenario-analytic approach.

Health-economic modelling is useful for assessing the clinical requirements and impact of new vaccines. In this study, we estimate the impact of potential vaccination for respiratory syncytial virus (RSV) of infants in the Netherlands. A decision analysis model was employed using seasonal data from a cohort of children (1996-1997 through 1999-2000) to assess hospitalisation, costs and impact of vaccination. Yearly, an estimated 3670 infants are hospitalised with RSV-infection in the Netherlands, vaccination protecting infants from 3 months of life onwards could prevent approximately 1000-3000 hospitalisations, depending on the effectiveness of the potential vaccine. Additionally, vaccination could prevent a major share of RSV-related costs. Comparison of the calculated break-even prices with the average price of recently introduced vaccines indicates that pricing for a potential RSV-vaccine most likely allows for only a single dose vaccination or several doses at a relatively low price per dose in order to achieve cost savings. However, if evidence on relevant RSV-related mortality would become available, higher pricing would be justified, while still remaining below accepted thresholds for cost-effectiveness.

Cardiac overexpression of human VEGF(165) by recombinant Semliki Forest virus leads to adverse effects in pressure-induced heart failure.

Semliki Forest virus (SFV) is an efficient vector for cardiac gene delivery. The relatively short transgene expression induced by SFV seems appropriate for angiogenic gene therapy. We tested the effects of SFV expressing vascular endothelial growth factor (VEGF) on cardiac angiogenesis and heart failure in the mRen2 transgenic rat.Six-week-old mRen2 rats received SFV-VEGF or control virus (n=7 each) administered intracoronarily. Twelve days after transfection, cardiac capillary density and function were assessed. Capillary density in cardiac regions where SFV expression was highest had decreased by 20% in the SFV-VEGF-treated group. The decrease in capillary density was accompanied by impaired systolic function as illustrated by increased endsystolic volumes and a 34% decrease in cardiac output.We conclude that the time frame of SFV expression is sufficient to induce structural alterations, but that VEGF in mRen2 transgenic rats did not elicit the expected angiogenic effect. Rather, capillary density was decreased and subsequently cardiac function was impaired. This paradoxical finding is possibly related to the pathophysiology associated with this model and warrants caution if one is to pursue VEGF-mediated, angiogenic therapy before proceeding to a clinical setting. (Neth Heart J 2007;15:335-41.).

The involvement of the lipid phase transition in the plasma-induced dissolution of multilamellar phosphatidylcholine vesicles.

Unsonicated liposomes prepared from dimyristoyl phosphatidylcholine were nearly completely dissolved during a 3 h incubation with rat plasma at or close to the phase-transition temperature of 24 degrees C. At 37 or 15 degrees C virtually no liposomal disintegration was observed even after 24 h of incubation. The liposomal solubilization, which was monitored by turbidity measurements or by determination of phospholipid sedimentability, was accompanied by the formation of a phospholipid-protein complex similar or identical to the one we previously reported to be formed from sonicated liposomes of egg phosphatidylcholine (Scherphof, G., Roerdink, F., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296--307). Unsonicated multilamellar liposomes made of egg phosphatidylcholine were completely resistant to the dissolving potency of plasma when incubated at 37 degrees C. Liposomes from equimolar mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine were only degraded by plasma in the temperature range between 30 and 35 degrees C at which temperature this cocrystallizing phospholipid mixture undergoes a phase transition. However, even at these temperatures the rate of dissolution of this mixture was significantly lower than of dimyristoyl phosphatidylcholine at 24 degrees C. In the dissolving process of this mixture a slight preference for the lower-melting component was observed. The ability of cholesterol to completely abolish the susceptibility of dimyristoyl phosphatidylcholine liposomes to plasma at a 1:2 molar ratio of cholesterol to phospholipid substantiates the essential role of the phase transition in the process of liposome solubilization. When liposomes of the monotectic mixtures dimyristoyl and distearoyl phosphatidylcholine or dilauroyl and distearoyl phosphatidylcholine were incubated with plasma at temperatures in between those at which the constituent lipids undergo a phase change in the mixture, the liposomes were slowly dissolved. Under those conditions a selective removal of the lipids in the liquid-crystalline phase was observed. It is concluded that for the plasma-induced dissolution of unsonicated liposomes, which is most probably achieved by interaction with (apo)lipoproteins, the presence of phase boundaries is required in much the same way as was first reported for phospholipases by Op den Kamp, J.A.F., de Gier, J. and Van Deenen, L.L.M. (1974) Biochim. Biophys. Acta 345, 253--256).

Action of phospholipases A2 on phosphatidylcholine bilayers. Effects of the phase transition, bilayer curvature and structural defects.

We examined the action of porcine pancreatic and bee-venom phospholipase A2 towards bilayers of phosphatidylcholine as a function of several physical characteristics of the lipid-water interface. 1. Unsonicated liposomes of dimyristoyl phosphatidylcholine are degraded by both phospholipases in the temperature region of the phase transition only (cf. Op den Kamp et al. (1974) Biochim. Biophys. Acta 345, 253--256 and Op den Kamp et al. (1975) Biochim. Biophys. Acta 406, 169--177). With sonicates the temperature range in which hydrolysis occurs is much wider. This discrepancy between liposomes and sonicates cannot be ascribed entirely to differences in available substrate surface. 2. Below the phase-transition temperature the phospholipases degrade dimyristoyl phosphatidylcholine single-bilayer vesicles with a strongly curved surface much more effectively than larger single-bilayer vesicles with a relatively low degree of curvature. 3. Vesicles composed of egg phosphatidylcholine can be degraded by pancreatic phospholipase A2 at 37 degrees C, provided that the substrate bilayer is strongly curved. The bee-venom enzyme shows a similar, but less pronounced, preference for small substrate vesicles. 4. In a limited temperature region just above the transition temperature of the substrate the action of both phospholipases initially proceeds with a gradually increasing velocity. This stimulation is presumably due to an increase of the transition temperature, effectuated by the products of the phospholipase action. 5. Structural defects in the substrate bilayer, introduced by sonication below the phase-transition temperature (cf. Lawaczeck et al. (1976) Biochim. Biophys. Acta 443, 313--330) facilitate the action of both phospholipases. The results lead to the general conclusion that structural irregularities in the packing of the substrate molecules facilitate the action of phospholipases A2 on phosphatidylcholine bilayers. Within the phase transition and with bilayers containing structural defects these irregularities represent boundaries between separate lipid domains. The stimulatory effect of strong bilayer curvature can be ascribed to an overall perturbation of the lipid packing as well as to a change in the phase-transition temperature.

Hydrolysis of phosphatidylcholine liposomes by phospholipases A2. Effects of the local anesthetic dibucaine.

(1) Dibucaine evokes a downward shift in the phase transition temperature of saturated phosphatidylcholines, while it also affects the pretransition. (2) The binding of dibucaine to phosphatidylcholine liposomes increases sharply when the lipid is transformed from the gel phase to the liquid-crystalline phase. (3) The activity of Naja naja phospholipase A2 towards dimyristoyl phosphatidylcholine liposomes is either stimulated or inhibited by dibucaine, depending on whether the substrate is in the gel or the liquid-crystalline state, respectively, whereas the activity of pancreatic phospholipase A2 is inhibited by the anesthetic irrespective of the physical state of the substrate. This observation is further substantiated by the results of studies on liposomes prepared from mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine or dilauroyl and distearoyl phosphatidylcholine. (4) The uptake of dibucaine by positively charged liposomes composed of phosphatidylcholine and stearylamine is considerably reduced in comparison with pure phosphatidylcholine liposomes. This decrease is paralleled by a reduction of the inhibitory and stimulatory effects of dibucaine on the hydrolysis of such liposomes by pancreatic and Naja naja phospholipase, respectively. (5) The inhibitory action of dibucaine towards the pancreatic phospholipase is lowered by increasing CaCl2 concentrations. This reduction is accompanied by a decreased uptake of anesthetic by the liposomes.