Emanuele Montomoli

A Clinical Trial of a Whole-Virus H5N1 Vaccine Derived from Cell Culture

BACKGROUND Widespread infections of avian species with avian influenza H5N1 virus and its limited spread to humans suggest that the virus has the potential to cause a human influenza pandemic. An urgent need exists for an H5N1 vaccine that is effective against divergent strains of H5N1 virus. METHODS In a randomized, dose-escalation, phase 1 and 2 study involving six subgroups, we investigated the safety of an H5N1 whole-virus vaccine produced on Vero cell cultures and determined its ability to induce antibodies capable of neutralizing various H5N1 strains. In two visits 21 days apart, 275 volunteers between the ages of 18 and 45 years received two doses of vaccine that each contained 3.75 microg, 7.5 microg, 15 microg, or 30 microg of hemagglutinin antigen with alum adjuvant or 7.5 microg or 15 microg of hemagglutinin antigen without adjuvant. Serologic analysis was performed at baseline and on days 21 and 42. RESULTS The vaccine induced a neutralizing immune response not only against the clade 1 (A/Vietnam/1203/2004) virus strain but also against the clade 2 and 3 strains. The use of adjuvants did not improve the antibody response. Maximum responses to the vaccine strain were obtained with formulations containing 7.5 microg and 15 microg of hemagglutinin antigen without adjuvant. Mild pain at the injection site (in 9 to 27% of subjects) and headache (in 6 to 31% of subjects) were the most common adverse events identified for all vaccine formulations. CONCLUSIONS A two-dose vaccine regimen of either 7.5 microg or 15 microg of hemagglutinin antigen without adjuvant induced neutralizing antibodies against diverse H5N1 virus strains in a high percentage of subjects, suggesting that this may be a useful H5N1 vaccine. (ClinicalTrials.gov number, NCT00349141.)

Fast rise of broadly cross-reactive antibodies after boosting long-lived human memory B cells primed by an MF59 adjuvanted prepandemic vaccine

Proactive priming before the next pandemic could induce immune memory responses to novel influenza antigens. In an open-label study, we analyzed B cell memory and antibody responses of 54 adults who received 2 7.5-μg doses of MF59-adjuvanted A/Vietnam/1194/2004 clade 1 (H5N1) vaccine. Twenty-four subjects had been previously primed with MF59-adjuvanted or plain clade 0-like A/duck/Singapore/1997 (H5N3) vaccine during 1999–2001. The prevaccination frequency of circulating memory B cells reactive to A/Vietnam/1194/2004 was low in both primed and unprimed individuals. However, at day 21 after boosting, MF59-adjuvanted primed subjects displayed a higher frequency of H5N1-specific memory B cells than plain-primed or unprimed subjects. The immune memory was rapidly mobilized by a single vaccine administration and resulted in high titers of neutralizing antibodies to antigenically diverse clade 0, 1, and 2 H5N1 viruses already at day 7. In general, postvaccination antibody titers were significantly higher in primed subjects than in unprimed subjects. Subjects primed with MF59-adjuvanted vaccine responded significantly better than those primed with plain vaccine, most notably in early induction and duration of cross-reacting antibody responses. After 6 months, high titers of cross-reactive antibody remained detectable among MF59-primed subjects. We conclude that distant priming with clade 0-like H5N3 induces a pool of cross-reactive memory B cells that can be boosted rapidly years afterward by a mismatched MF59-adjuvanted vaccine to generate high titers of cross-reactive neutralizing antibodies rapidly. These results suggest that pre-pandemic vaccination strategies should be considered.

Preclinical and Clinical Development of Plant-Made Virus-Like Particle Vaccine against Avian H5N1 Influenza

The recent swine H1N1 influenza outbreak demonstrated that egg-based vaccine manufacturing has an Achilles heel: its inability to provide a large number of doses quickly. Using a novel manufacturing platform based on transient expression of influenza surface glycoproteins in Nicotiana benthamiana, we have recently demonstrated that a candidate Virus-Like Particle (VLP) vaccine can be generated within 3 weeks of release of sequence information. Herein we report that alum-adjuvanted plant-made VLPs containing the hemagglutinin (HA) protein of H5N1 influenza (A/Indonesia/5/05) can induce cross-reactive antibodies in ferrets. Even low doses of this vaccine prevented pathology and reduced viral loads following heterotypic lethal challenge. We further report on safety and immunogenicity from a Phase I clinical study of the plant-made H5 VLP vaccine in healthy adults 18–60 years of age who received 2 doses 21 days apart of 5, 10 or 20 µg of alum-adjuvanted H5 VLP vaccine or placebo (alum). The vaccine was well tolerated at all doses. Adverse events (AE) were mild-to-moderate and self-limited. Pain at the injection site was the most frequent AE, reported in 70% of vaccinated subjects versus 50% of the placebo recipients. No allergic reactions were reported and the plant-made vaccine did not significantly increase the level of naturally occurring serum antibodies to plant-specific sugar moieties. The immunogenicity of the H5 VLP vaccine was evaluated by Hemagglutination-Inhibition (HI), Single Radial Hemolysis (SRH) and MicroNeutralisation (MN). Results from these three assays were highly correlated and showed similar trends across doses. There was a clear dose-response in all measures of immunogenicity and almost 96% of those in the higher dose groups (2×10 or 20 µg) mounted detectable MN responses. Evidence of striking cross-protection in ferrets combined with a good safety profile and promising immunogenicity in humans suggest that plant-based VLP vaccines should be further evaluated for use in pre-pandemic or pandemic situations. Trial Registration ClinicalTrials.gov NCT00984945

MF59®-Adjuvanted H5N1 Vaccine Induces Immunologic Memory and Heterotypic Antibody Responses in Non-Elderly and Elderly Adults

Background Pathogenic avian influenza virus (H5N1) has the potential to cause a major global pandemic in humans. Safe and effective vaccines that induce immunologic memory and broad heterotypic response are needed. Methods and Findings Healthy adults aged 18–60 and >60 years (n = 313 and n = 173, respectively) were randomized (1∶1) to receive two primary and one booster injection of 7.5 μg or 15 μg doses of a subunit MF59-adjuvanted H5N1 (A/Vietnam/1194/2004) (clade 1) vaccine. Safety was monitored until 6 months after booster. Immunogenicity was assessed by hemagglutination inhibition (HI), single radial hemolysis (SRH) and microneutralization assays (MN). Mild injection-site pain was the most common adverse reaction. No serious adverse events relating to the vaccine were reported. The humoral immune responses to 7.5 μg and 15 μg doses were comparable. The rates for seroprotection (HI>40; SRH>25mm2; MN ≥40) after the primary vaccination ranged 72–87%. Six months after primary vaccination with the 7.5 μg dose, 18% and 21% of non-elderly and elderly adults were seroprotected; rates increased to 90% and 84%, respectively, after the booster vaccination. In the 15 μg group, seroprotection rates among non-elderly and elderly adults increased from 25% and 62% after primary vaccination to 92% and 88% after booster vaccination, respectively. A heterologous immune response to the H5N1/turkey/Turkey/05 strain was elicited after second and booster vaccinations. Conclusions Both formulations of MF59-adjuvanted influenza H5N1 vaccine were well tolerated. The European Union requirement for licensure for pre-pandemic vaccines was met by the lower dose tested. The presence of cross-reactive antibodies to a clade 2 heterologous strain demonstrates that this vaccine may be appropriate for pre-pandemic programs. Trial Registration ClinicalTrials.gov NCT00311480

Antibodies to influenza nucleoprotein cross-react with human hypocretin receptor 2.

Similarity between influenza nucleoprotein and hypocretin receptor 2 may trigger vaccine-associated narcolepsy. Immunological mistaken identity New reports of narcolepsy increased after the vaccination campaign against the 2009 A(H1N1) influenza pandemic in some countries but not others. Now Ahmed et al. examine differences between the vaccines used and find a potential mechanistic explanation for the vaccine-specific effect. They found a peptide in influenza nucleopeptide A that shared protein residues with human hypocretin receptor 2, which has been linked to narcolepsy. The vaccine used in unaffected countries contained less influenza nucleoprotein. Indeed, patients with putative vaccine-associated narcolepsy produced antibodies that cross-reacted to both the influenza and the hypocretin receptor 2 epitopes. Although these data do not demonstrate causation, they provide a possible explanation for the association of this particular influenza vaccination with increased reports of narcolepsy. The sleep disorder narcolepsy is linked to the HLA-DQB1*0602 haplotype and dysregulation of the hypocretin ligand-hypocretin receptor pathway. Narcolepsy was associated with Pandemrix vaccination (an adjuvanted, influenza pandemic vaccine) and also with infection by influenza virus during the 2009 A(H1N1) influenza pandemic. In contrast, very few cases were reported after Focetria vaccination (a differently manufactured adjuvanted influenza pandemic vaccine). We hypothesized that differences between these vaccines (which are derived from inactivated influenza viral proteins) explain the association of narcolepsy with Pandemrix-vaccinated subjects. A mimic peptide was identified from a surface-exposed region of influenza nucleoprotein A that shared protein residues in common with a fragment of the first extracellular domain of hypocretin receptor 2. A significant proportion of sera from HLA-DQB1*0602 haplotype–positive narcoleptic Finnish patients with a history of Pandemrix vaccination (vaccine-associated narcolepsy) contained antibodies to hypocretin receptor 2 compared to sera from nonnarcoleptic individuals with either 2009 A(H1N1) pandemic influenza infection or history of Focetria vaccination. Antibodies from vaccine-associated narcolepsy sera cross-reacted with both influenza nucleoprotein and hypocretin receptor 2, which was demonstrated by competitive binding using 21-mer peptide (containing the identified nucleoprotein mimic) and 55-mer recombinant peptide (first extracellular domain of hypocretin receptor 2) on cell lines expressing human hypocretin receptor 2. Mass spectrometry indicated that relative to Pandemrix, Focetria contained 72.7% less influenza nucleoprotein. In accord, no durable antibody responses to nucleoprotein were detected in sera from Focetria-vaccinated nonnarcoleptic subjects. Thus, differences in vaccine nucleoprotein content and respective immune response may explain the narcolepsy association with Pandemrix.

Increased immunogenicity of the MF59-adjuvanted influenza vaccine compared to a conventional subunit vaccine in elderly subjects.

Three-hundred and eight outpatient elderly subjects (≥ 65 years) were randomly assigned to receive the MF59-adjuvanted influenza vaccine (FLUAD; n = 204) or a conventional subunit influenza vaccine (AGRIPPAL S1; n = 104) in order to compare the safety and immunogenicity of the two vaccines. Although mild pain at the injection site was reported more frequently by subjects immunised with the adjuvanted vaccine, both vaccines were shown to be safe and well tolerated. The adjuvanted vaccine was more immunogenic as indicated by higher post-immunisation geometric mean titres (GMTs) and by higher proportions of subjects with post-immunisation ≥ four fold increases of antibody titres or subjects with ≥ 1/160 post-immunisation HI titres. These differences, statistically significant for all three strains after immunisation, indicated that, by addition of the MF59 adjuvant emulsion, conventional subunit influenza antigens acquire an enhanced immunogenicity without any clinically significant increase of their reactogenicity.

Cross-protection by MF59-adjuvanted influenza vaccine: neutralizing and haemagglutination-inhibiting antibody activity against A(H3N2) drifted influenza viruses.

Adjuvants enhance antibody response against vaccination. We compared the ability of MF59-adjuvanted and non-adjuvanted subunit influenza vaccines, containing A/Wyoming/3/03(H3N2), to confer cross-protection against four consecutive drifted strains in the elderly. Neutralizing and haemagglutination-inhibiting antibody were measured. MF59-adjuvanted vaccine induced a stronger booster response against A/Panama/2007/99(H3N2) than non-adjuvanted vaccine. A/Panama/2007/99(H3N2) circulated widely during the previous 5 years and was included in vaccines over four consecutive seasons. Broader serological protection against drifted strains that circulated 1 and 2 years after vaccination with A/Wyoming/3/03(H3N2) was observed with MF59-adjuvanted vaccine. Thus, MF59-adjuvanted vaccine confers greater immunogenicity than non-adjuvanted vaccines in vulnerable populations.

Cross-reactive antibody responses to the 2009 A/H1N1v influenza virus in the Italian population in the pre-pandemic period

To assess in Italy the pre-pandemic susceptibility of the general population to the 2009 A/H1N1v influenza virus, 587 serum samples collected in 2004 were analyzed using haemagglutination-inhibition (HI), single-radial-haemolysis (SRH) and microneutralisation (MN) assays. Serum samples were stratified by age group, gender, and geographic area. Overall, using HI assay, the proportion of subjects showing antibodies cross-reacting with 2009 A/H1N1v virus at seroprotection level (>or=1:40) was estimated to be 6.7%, 12.4%, and 22.4% in individuals born between 2004 and 1949, 1948 and 1939, 1938 and 1909, respectively. With a HI antibody titre of >or=1:10, in the same birth cohort, the seroprotection levels were 13.5%, 19.2%, and 58.2%, respectively. The results suggest that the Italian population was not fully naïf to the current pandemic virus and that the possible previous exposure and immune response increases with age.

Current adjuvants and new perspectives in vaccine formulation

Given the important role of adjuvants in prophylactic vaccines, identification and development of new adjuvants with enhanced efficacy and safety is necessary. The use of adjuvants with immunopotentiating properties that can direct the immune responses to humoral or cell-mediated immunity and can induce T-cell responses has made it possible to design more protective vaccines. Although current regulations focus on traditional adjuvants, notably aluminum and calcium salts, advances have been made in regulatory considerations. The regulatory agencies for the evaluation of medicinal products are actively drafting guidance on requirements for the evaluation of new adjuvants. This article briefly summarizes the most widely studied adjuvants in vaccination, including those licensed for human vaccines and the regulatory aspects relevant to adjuvant quality at development stages.

Immunogenicity and Safety of MF59-Adjuvanted H5N1 Influenza Vaccine From Infancy to Adolescence

OBJECTIVE: This study evaluated the immunogenicity, safety, and tolerability of a MF59-adjuvanted H5N1 vaccine in a population 6 months through 17 years of age. METHODS: Healthy subjects 6 to <36 months, 3 to <9 months, and 9 to <18 years of age were assigned randomly to receive 2 doses of either a MF59-adjuvanted H5N1 vaccine (7.5 μg/dose) or a MF59-adjuvanted trivalent seasonal influenza control vaccine (15 μg/dose for each antigen). Immunogenicity against the A/Vietnam/1194/2004-like vaccine strain was measured before and 3 weeks after the 2-dose primary series, through hemagglutination inhibition (HI), single radial hemolysis (SRH), and microneutralization. Local and systemic reactions were recorded. RESULTS: A total of 335 subjects received the H5N1 vaccine, and 137 subjects received the seasonal vaccine. Rates of seroprotection (HI titer of ≥40) against the H5N1 vaccine antigen were 97% for children 6 to 36 months and 3 to 9 years of age and 89% for older children. All subjects seroconverted in the SRH assay. Microneutralization titers of ≥40 were achieved by 99% of subjects, and ≥98% of subjects, respectively. Local reactions, particularly injection site pain in older children, were common, generally mild to moderate in nature, and transient and resolved spontaneously. Up to 5% of participants. There were no vaccine-related serious adverse events in either group. CONCLUSIONS: In this pediatric population, MF59-adjuvanted H5N1 vaccine was highly immunogenic, had a good safety profile, reactogenicity comparable with that of an adjuvanted seasonal influenza control vaccine.