Virosomal Influenza Vaccine Research Articles

Preventing influenza coinfection among HIV-infected persons: a complex picture coming into focus

Preventing influenza coinfection among HIV-infected persons: a complex picture coming into focus

Virosomal influenza-vaccine induced immunity in HIV-infected individuals with high versus low CD4+ T-cell counts: clues towards a rational vaccination strategy

In a prospective influenza-vaccination trial we show that HIV-infected individuals with CD4 T-cell counts less than 350 microl were distinct from HIV-infected individuals with more than 350 CD4 T-cell counts/microl, and from HIV-negative individuals, in that an influenza-specific immunoglobulin M-response was absent and expansion of interferon-gamma-secreting CD4 T cells was impaired. By contrast, immunoglobulin G-responses were induced in all study groups. These data suggest that establishing broad influenza-specific (immunoglobulin G) B-cell memory prior to severe immunodeficiency is important.

Safety of a Virosomal Adjuvanted Influenza Vaccine in Children Suffering from Chronic Disease~!2009-08-17~!2010-02-23~!2010-04-20~!

Children suffering from chronic disease are at increased risk for complications and mortality caused by influenza. Influenza can be most effectively prevented by vaccination. Although recommended for children with chronic disease, vaccination rates remain low in this population. One possible explanation is the parents' concern that the child's underlying disease may worsen or that adverse events following influenza vaccination may be more severe than in healthy children. Data demonstrating the safety of influenza vaccination in this population are therefore crucial and may help to increase future vaccination rates in children suffering from chronic disease. The present review summarises published data on safety following vaccination with a virosomal adjuvanted influenza vaccine in children suffering from chronic disease compared with healthy children. The vaccine was shown to be immunogenic and safe in four particularly vulner- able populations: children suffering from asthma, cystic fibrosis, diabetes, or HIV infection. No changes in the course of the underlying diseases were observed after vaccination.

Utility of virosomal adjuvated influenza vaccines: a review of the literature.

In spite of the efforts of the World Health Organization (WHO), influenza continues to be a major public health problem, both because of its impact on the health of subjects at risk, such as the elderly, and because of the economic burden that it places on society. Adjuvants are agents which, when incorporated into vaccines, enhance the immunogenicity of their antigens. The need for ever more immunogenic and efficacious influenza vaccines has led to the development of innovative vaccines. One of these, the virosomal vaccine, has been on the market since 1997. The results obtained through controlled clinical studies and widespread application in the field suggest that the virosomal vaccine is not only an important tool for the prevention of seasonal influenza but also a valid means of potentiating the effect of a pandemic influenza vaccine and, perhaps, of preparing multivalent or combined vaccines.

The virosomal adjuvanted influenza vaccine

Importance of the field: The protection conferred by influenza vaccines varies for several reasons, for example the age or degree of immune depression of the recipient. All currently available seasonal influenza vaccines are safe and substantially effective in preventing influenza in healthy people. However, elderly people and patients with chronic diseases or immune system defects need a more effective vaccine to avoid serious risks from influenza and its complications. Research has been undertaken to improve the efficacy of vaccination. Recent research includes the use of new adjuvants or antigen-presenting strategies.Areas covered in this review: The virosomal adjuvanted subunit influenza vaccine has been studied in groups for whom vaccination is recommended. We describe virosomal technology, including production and mode of action, as well as the available efficacy, immunogenicity and safety data, with the aim of understanding the benefits of this vaccine's use.What the reader will gain: A review of published data on efficacy, immunogenicity and safety from sponsor- and investigator- driven studies, focusing on recent publications.Take home message: The vaccine was generally very immunogenic and safe in all investigated populations. Its ability to induce protective antibody titers has been shown to exceed that of conventional influenza vaccines in elderly people and individuals with little or no prior exposure to the viral strains.

Immunogenicity and tolerability of a virosome influenza vaccine compared to split influenza vaccine in patients with sickle cell anemia

The immunogenicity and tolerability of virosome and of split influenza vaccines in patients with sickle cell anemia (SS) were evaluated. Ninety SS patients from 8 to 34 years old were randomly assigned to receive either virosome (n=43) or split vaccine (n=47). Two blood samples were collected, one before and one 4–6 weeks after vaccination. Antibodies against viral strains (2006) A/New Caledonia (H1N1), A/California (H3N2), B/Malaysia were determined using the hemagglutinin inhibition test. Post-vaccine reactions were recorded over 7 days. Seroconversion rates for H1N1, H3N2 and B were 65.1%, 60.4% and 83.7% for virosome vaccine, and 68.0%, 61.7% and 68.0% for split vaccine. Seroprotection rates for H1N1, H3N2 e B were 100%, 97.6% and 69.7% for virosome, and 97.8%, 97.8% and 76.6% for split vaccine. No severe adverse reactions were recorded. Virosome and split vaccines in patients with sickle cell anemia were equally immunogenic, with high seroconversion and seroprotection rates. Both vaccines were well tolerated.

Low reactogenicity of the virosomal subunit influenza vaccine in healthy children without risk factors

ObjectivesTo estimate the reactogenicity of the inactivated virosomal subunit influenza vaccine in healthy children. Material and methodsDuring the 2004-2005 influenza season we carried out a prospective cohort study in 11 paediatric clinics of the metropolitan region of Barcelona to evaluate the effectiveness, efficiency and reactogenicity of one dose of inactivated virosomal subunit influenza vaccine in healthy children. The vaccine was administered to healthy children aged 3-14 years attended in 5 of the 11 clinics whose parents gave informed consent to participate in the study. These children formed the vaccinated cohort (n=966). The unvaccinated cohort (n=985) was formed by children attended in the other six clinics whose parents agreed to participate. ResultsSeven point five percent of the 966 children vaccinated suffered one or more local or systemic adverse reactions. The most common were local reactions: erythema (4.04%) and induration (1.76%). Systemic reactions were much less frequent, with malaise (0.72%) and fever (0.52%) the most common. ConclusionsThe virosomal influenza vaccine has low levels of reactogenicity and is very well tolerated in healthy children.

Swine flu vaccination in patients with egg allergy

Swine flu, a new subtype of influenza A virus H1N1, not previously detected in pigs or humans has arrived in the United Kingdom in recent months with hundreds and thousands of cases already confirmed in both adults and children. The World Health Organisation (WHO) has raised the threat level to Phase 6 (pandemic) to reflect the ongoing community outbreaks of swine flu throughout the world. So far in the United Kingdom, most cases have been mild but clustering of cases shows infectivity and with an anticipated increase in cases towards the autumn, a potential winter crisis looms. International data show the age profile of affected subjects is predominantly in children especially young children and young adults up to age 30 years. The WHO has reported that fatalities have occurred in pregnant women, those with significant comorbidities and also in previously healthy young individuals. The Department of Health (DoH) is planning a mass vaccination programme as soon as the pharmaceutical industry produces sufficient quantities of an effective vaccine. Swine flu vaccine is likely to be manufactured in a similar manner to the currently available influenza vaccines by inoculating embryonated chicken eggs with the virus. The vaccine will then be extracted from the extraembryonic fluid by chemically inactivating the virus that is then further treated and purified. Consequently influenza vaccines contain measurable quantities of egg protein [1] with both fatal and non-fatal anaphylaxis reported following administration [2, 3]. Therefore, there is a potential risk of allergic reaction for the 1–2% of children who have egg allergy [4] and a significant number of adults who have either persisting or adult-onset egg allergy. Each year the European Pharmacopoeia publishes guidance on the maximum allowable ovalbumin content of influenza vaccines. Up to the end of 2005, conventional split and subunit influenza vaccines were allowed to contain a maximum of 1mg ovalbumin per dose, whereas virosomal influenza vaccines were permittedo 50 ng/dose. In an apparent retrograde step, however, from 2006 all influenza vaccines were permitted a maximum ovalbumin content of up to 1mg/dose [5, 6]. Therefore, virosomal vaccines can no longer be relied upon to contain safe quantities of egg protein for the egg-allergic population. Furthermore, there is considerable variation in egg protein content between different brands of influenza vaccine and even between batches of the same vaccine [7]. Therefore, recommendations based on vaccine type that are applicable to the total egg-allergic population are not possible.

Long-lasting immunogenicity of a virosomal vaccine in older children and young adults with type I diabetes mellitus

To evaluate the long-lasting immunogenicity and reactogenicity of a virosomal influenza vaccine in subjects with type I diabetes, a trial was conducted during the 2007–2008 influenza season in Milan, Northern Italy. One hundred five subjects aged 9–30 years were randomized to receive by intramuscular injection vaccination by a single dose (0.5 ml) of either a virosomal (Inflexal V ®) ( n = 52) or a standard subunit (Influvac ®) ( n = 53) vaccine. Serum hemagglutinin inhibition antibody titres were determined against the three recommended influenza-like strains, A/H1N1, A/H3N2 and B, at pre-vaccination, and 1 and 6 months post-vaccination. Geometric mean titres were increased in the two groups 1 and 6 months post-vaccination ( P < 0.001). One month post-vaccination both vaccines met the CPMP requirement for immunogenicity with high seroprotection rates (>95%) for strains A/H1N1 and A/H3N2, and a seroprotection of 73% and 70% in the virosomal and subunit vaccine for strain B. Mean fold increase ranged 2.8 (A/H3N2)–6.2 (A/H1N1) in the virosomal group and 2.3 (A/H3N2)–4.8 (A/H1N1) in the subunit group. Immunogenicity declined 6 months post-vaccination in both groups, and the CPMP requirement for immunogenicity was satisfied only in the virosomal group. In subjects without pre-existing antibodies to strain B (titre <10), the virosomal vaccine showed higher immune response than the subunit vaccine 6 months post-vaccination, with a geometric mean titre (95% CI) of 40.2 (30.7–54.6) vs. 21.2 (14.6–30.8). Reactogenicity was similar in the two vaccines. All reactions were transient and not severe. The results indicate that in older children and young adults with type I diabetes influenza vaccination with a virosomal or a standard subunit vaccine is safe and adequately immunogenic against the three influenza vaccine strains. In addition, the virosomal vaccine may show better long-lasting immune response than the standard subunit vaccine, especially in subjects without pre-existing antibodies to influenza strains.

Randomized, Double‐Blind Comparative Trial of Subunit and Virosomal Influenza Vaccines for Immunocompromised Patients

To our knowledge, no study to date has compared the effects of a subunit influenza vaccine with those of a virosomal influenza vaccine on immunocompromised patients. A prospective, double-blind, randomized study was conducted to compare the immunogenicity and reactogenicity of subunit and virosomal influenza vaccines for adult patients who had an immunosuppressive disease or who were immunocompromised as a result of treatment. There were 304 patients enrolled in our study: 131 with human immunodeficiency virus (HIV) infection, 47 with a chronic rheumatologic disease, 74 who underwent a renal transplant, 47 who received long-term hemodialysis, and 5 who had some other nephrologic disease. There were 151 patients who received the subunit vaccine and 153 patients who received the virosomal vaccine. A slightly higher percentage of patients from the subunit vaccine group were protected against all 3 influenza vaccine strains after being vaccinated, compared with patients from the virosomal vaccine group (41% vs. 30% of patients; P = .03). Among HIV-infected patients, the level of HIV RNA, but not the CD4 cell count, was an independent predictor of vaccine response. Among renal transplant patients, treatment with mycophenolate significantly reduced the immune response to vaccination. The 2 vaccines were comparable with regard to the frequency and severity of local and systemic reactions within 7 days after vaccination. Disease-specific scores for the activity of rheumatologic diseases did not indicate flare-ups 4-6 weeks after vaccination. For immunosuppressed patients, the subunit vaccine was slightly more immunogenic than the virosomal vaccine. The 2 vaccines were comparable with regard to reactogenicity. Vaccine response decreased with increasing degree of immune suppression. Among HIV-infected patients, the viral load, rather than the CD4 cell count, predicted the protective immune response to the vaccine. NCT00783380 .

Immunogenicity and safety of low dose virosomal adjuvanted influenza vaccine administered intradermally compared to intramuscular full dose administration

Background Despite the established benefit of intramuscular (i.m.) influenza vaccination, new adjuvants and delivery methods for comparable or improved immunogenicity are being explored. Intradermal (i.d.) antigen administration is hypothesized to initiate an efficient immune response at reduced antigen doses similar to that observed after i.m. full dose vaccination. Methods In a randomized, partially blinded phase II study 224, healthy adults aged ≥18 to ≤60 years were randomly assigned to four groups and received trivalent influenza vaccine at single doses of 3.0, 4.5 and 6.0 μg hemagglutinin (HA) antigen of each influenza virus strain via i.d. injection or 15.0 μg HA of each influenza strain via i.m. delivery. Serum anti-influenza virus antibodies were determined by hemagglutination inhibition (HI) assay before and 3 weeks after vaccination. Safety assessments were made at baseline and at the follow-up visit by the investigators and for a 4-day period post-vaccination by the subjects themselves. Results The EMEA requirements for re-licensing of influenza vaccines were fulfilled in all groups 3 weeks after vaccination, irrespective of dose and mode of administration. High seroconversion rates were observed in all study groups and for all strains ranging from 50.9 to 85.5% and 70.4 to 87.0% after i.d. and i.m. vaccination, respectively. Seroprotection rates for the A strains A/Solomon Islands and A/Wisconsin were generally higher compared to the B/Malaysia strain and ranged from 89.1 to 98.2% across the i.d. groups. Similar rates of 96.3% for the A/Solomon Islands and 94.4% for the A/Wisconsin strain were observed in the i.m. group. Seroprotection rates for the B/Malaysia strain were 65.5, 83.0 and 72.7% after i.d. administration of 3.0, 4.5, and 6.0 μg HA of each strain, respectively, compared to a seroprotection rate of 85.2% in the i.m. group. In addition, marked increases in geometric mean titer (GMT) were observed across the groups for all influenza virus strains ranging from 6.9 to 70.5 for i.d. and from 16.9 to 56.5 for i.m. antigen delivery. Both routes of administration were well tolerated. Systemic reactions were broadly similar across the groups. With respect to local reactions the frequency of injection site pain and ecchymosis were significantly lower following i.d. vaccination, while other local reactions such as erythema occurred at higher rates with i.d. than with i.m. vaccine administration, as expected due to the mechanism of action for the i.d. route. Conclusions The virosomal adjuvanted influenza vaccine (Inflexal ®V) was shown to be overall highly immunogenic and well tolerated when given i.d. at reduced doses to healthy adults, eliciting an immune response similar to that observed with full dose i.m. administration and thus suggesting a promising antigen-sparing strategy for universal influenza vaccination against endemic influenza. Trial registration ISRCTN registry number: 33950739.

Economic benefits for the family of inactivated subunit virosomal influenza vaccination of healthy children aged 3–14 years during the annual health examination in private paediatric offices

Taking the results of a prospective cohort study by our group that evaluated the effectiveness of the inactivated subunit virosomal influenza vaccine (Inflexal V ®, Crucell-Berna) in the prevention of influenza-related diseases and the reduction of its negative economic consequences, the economic costs and benefits for the family of vaccinating a theoretical cohort of 1000 healthy children aged 3–14 years with no risk factors with one dose of vaccine during the yearly health examination were quantiified. The economic analysis was carried out from the family perspective and the time horizon of the study was established at 6 months. In the base case, the net present value was 21,551.62 euros (21.5 euros per vaccinated child), and the benefit–cost ratio was 2.15, meaning that 1.15 euros is saved per euro invested.

Safe vaccination of children with a virosomal adjuvanted influenza vaccine

A post-marketing study was conducted in 26 paediatric practices in Germany to collect safety information on the virosomal adjuvanted influenza vaccine Inflexal ® V (in Germany marketed as Infectovac Flu ®). Children aged 6 months to 6 years received one or two doses. Adverse events were documented by parents/legal guardians in a questionnaire for four days after vaccination. The rates of adverse events were low: 14% of vaccine exposures were associated with systemic and 25% with local adverse events. Most symptoms were mild to moderate and resolved within a few days. Nearly 100% of parents/legal guardians would agree to have their child vaccinated again with Inflexal ® V. Vaccination with Inflexal ® V was safe, well tolerated and highly accepted by parents/legal guardians.

Safe administration of an inactivated virosomal adjuvanted influenza vaccine in asthmatic children with egg allergy

In order to evaluate whether the virosomal adjuvanted influenza vaccine that has been shown to have the lowest egg protein content (Inflexal V, Berna Biotech) could be administered to children with even severe egg allergy without any risk of allergic reactions, we used epicutaneous skin testing with the undiluted vaccine in 88 asthmatic children (44 with and 44 without egg allergy), none of whom had a positive response. They were then vaccinated with the whole dose of Inflexal V intramuscularly in a one-dose protocol, and the occurrence of any immediate or delayed adverse events were actively monitored for 28 days. The results showed the safety of the administration, and demonstrated that Inflexal V can be safely given without performing a vaccine skin test in children with any kind of egg allergy.

Antibody and T-cell responses to a virosomal adjuvanted H9N2 avian influenza vaccine: Impact of distinct additional adjuvants

A highly efficacious vaccine is required to counteract a threat of an avian influenza pandemic. Increasing the potency of vaccines by adjuvation is essential not only to overcome generally low immunogenicity of pandemic strains, but also to allow dose sparing and as such to make it feasible to satisfy huge global production demands. In this study we evaluated the ability of four distinct adjuvants to further increase immune responses to a virosomal adjuvanted avian H9N2 influenza vaccine in mice. Currently registered adjuvants aluminium phosphate, aluminium hydroxide and MF59, as well as a novel promising adjuvant MATRIX-M were included in the study. Our results demonstrate that all adjuvants significantly increased the H9N2 haemagglutinin (HA) inhibition and ELISA antibody titers induced with the virosomal adjuvanted vaccine. The adjuvants exhibited different effect on the isotype of virus specific antibodies, with MATRIX-M inducing the most pronounced skewing to IgG2a, i.e. towards Th1 type of response. While the virosomal adjuvanted pandemic influenza vaccine efficiently induced CD4 + T-cell response, with no further increase upon adjuvation, the CD8 + T-cell responses induced with virosomal adjuvanted vaccine could be significantly improved upon additional adjuvation with MATRIX-M or MF59. All adjuvants demonstrated a dose sparing effect, i.e. in combination with the virosomal adjuvanted pandemic influenza vaccine they increased immune responses to comparable level independent of the tested vaccine dose. In conclusion, our results demonstrate that immune responses to a virosomal adjuvanted pandemic influenza vaccine can be further enhanced by add-on adjuvants, with MATRIX-M being overall the most potent adjuvant in combination with virosomes, followed by MF59 and finally aluminium-based adjuvants.

Antibody induction by virosomal, MF59-adjuvanted, or conventional influenza vaccines in the elderly

In a randomized, observer-blind, three-arm, parallel group, multi-centre trial including 386 elderly subjects in four countries, the immunogenicity and safety was studied of three different trivalent inactivated surface antigen (subunit) influenza vaccine types: a conventional subunit influenza vaccine (SIV, brand: Influvac and two newer vaccines: a MF59-adjuvanted subunit influenza vaccine (adSIV, brand: Fluad and a virosomal subunit influenza vaccine (vSIV, brand: Invivac. All vaccines were trivalent containing 15 microg hemagglutinin of each virus strain as recommended by the World Health Organization for the 2004-2005 season. The study was designed to demonstrate the serological non-inferiority of vSIV to both adSIV and SIV in elderly persons. The secondary objective was to investigate whether vSIV is superior to adSIV with respect to local reactogenicity. For all three vaccine strains, the post-vaccination geometric mean titres were comparable between SIV and adSIV and between vSIV and SIV. Seroprotection rates (i.e. percentages of subjects with a post-vaccination titre >or=40) varied between 84.1-100% indicating that the three vaccines all induced a strong antibody response. Local and systemic reactions were more frequently associated with adSIV (46 and 32%, respectively) than with vSIV or SIV ( approximately 20%). Vaccinations caused only little inconvenience as measured by questionnaire. In general, all vaccines were safe and well tolerated. In this trial, virosomal vaccine had similar immunogenicity to MF59-adjuvanted and conventional subunit vaccine and was considerably less reactogenic than the MF59-adjuvanted vaccine in the elderly.

Cost-effectiveness analysis of inactivated virosomal subunit influenza vaccination in children aged 3–14 years from the provider and societal perspectives

The costs and benefits of vaccinating a theoretical cohort of 1000 preschool and school age children (3–14 years) with one dose of inactivated virosomal subunit influenza vaccine in primary health care centers of the Catalan Health Service during the fall annual health examination were compared with the current strategy of no routine vaccination. The economic analysis was carried out from the provider perspective (cost-effectiveness analysis) and from the societal perspective (cost-effectiveness and cost–benefit analysis). The time horizon of the study was established at 6 months. In the base case (cost of vaccination of €9.425, cost of paediatric visit plus antibiotic and antipyretic treatment of €42.50, cost of 1 day of hospital stay of €454.25, cost of the work lost by the mother to take care of her ill child of €29.2 and cost of 1 year of quality adjusted life year lost of €10,662), the vaccination does not save money from the provider perspective (net present value = €−1460.51), but the cost-effectiveness ratios are very reasonable (€5.80 per episode of acute febrile respiratory process avoided and €18.26 per quality adjusted life year saved). From the societal perspective, the vaccination saves money (net present value = €+7587.03) and the benefit–cost ratio is 1.80, meaning that €0.80 is saved per euro invested. Our study shows that vaccination of children 3–14 years old with a single dose of inactivated subunit influenza vaccine in primary health care centers during the fall annual health examination provides socioeconomic benefits to the society in addition to substantial health benefits for the child.

Statistical Analysis of Influenza Vaccine Lot Consistency Studies

This paper presents an integrated statistical approach to the analysis of influenza vaccine lot consistency studies in which three lots are compared. The approach ensures that the overall Type I error rate (i.e., the probability of wrongly concluding that the lots are similar) is controlled. It is argued that the optimum efficacy measure is the geometric mean titer. The approach is demonstrated using data from a randomized, double-blind lot consistency study in which three consecutive production lots of Solvay Pharmaceutical' new, virosomal subunit influenza vaccine Invivac ® were compared.

Effectiveness of virosomal subunit influenza vaccine in preventing influenza-related illnesses and its social and economic consequences in children aged 3–14 years: A prospective cohort study

To evaluate the effectiveness of a virosomal subunit influenza vaccine in preventing influenza-related illnesses and its social and economic consequences in children aged 3–14 years, a prospective cohort study was carried out during the 2004–2005 influenza season in 11 private pediatric clinics in the Barcelona metropolitan area. One dose of a virosomal subunit inactivated influenza vaccine (Inflexal V ® Berna) was given during September and October 2004 to healthy children aged 3–14 years attended in 5 of the 11 clinics. Who comprised the vaccinated cohort ( n = 966). The non-vaccinated cohort ( n = 985) was comprised of children attended in the other six clinics. Informed consent was obtained from all parents. The follow up was performed between 1 November 2004 and 31 March 2005. Using a self-administered questionnaire, information was collected from parents or guardians on any type of acute, febrile respiratory illness suffered by their children during the study period, including antibiotic use, and absence from school or work-loss of parents as a result of the illness. RT-PCR (influenza A + B + C) was carried out on pharyngeal and nasal samples obtained from children attended by pediatricians during this period in these clinics with the following symptoms: fever ≥ 38.5° lasting at least 72 h, cough or sore throat (influenza-like illness). Adjusted vaccination effectiveness was 58.6% (95% CI 49.2–66.3) in preventing acute febrile respiratory illnesses, 75.1% (95% CI 61.0–84.1) in preventing cases of influenza-like illnesses and 88.4% (95% CI 49.2–97.3) in preventing laboratory-confirmed cases of influenza A. The adjusted vaccination effectiveness in reducing antibiotic use (18.6%, 95% CI −4.2 to 3.64), absence from school (57.8%, 95% CI 47.9–65.9) and work-loss of parents (33.3%, 95% CI 8.9–51.2) in children affected by an acute febrile respiratory illness was somewhat lower. Vaccination of children aged 3–14 years in pediatric practices with one dose of virosomal subunit inactivated influenza vaccine has the potential to considerably reduce the health and social burdens caused by influenza-related illnesses.

The virosomal influenza vaccine Invivac ®: Immunogenicity and tolerability compared to an adjuvanted influenza vaccine (Fluad ®) in elderly subjects

Several approaches are currently being pursued in order to improve the efficacy of influenza vaccines in elderly individuals and others who have impaired immune responses to conventional influenza vaccines. There are two influenza vaccines available for elderly subjects: Fluad ® (Chiron) and Invivac ® (Solvay Pharmaceuticals). The present clinical study was a randomized, endpoint-blind, parallel group study in elderly subjects aged 61 years and older to investigate the safety and immunogenicity of these vaccines as compared to a standard influenza vaccine Invivac ® (Solvay Pharmaceuticals). The three vaccines had similar immunogenicity results, whereas the tolerability profile of Invivac was better as compared to Fluad.