Intanza(®) 15 mcg intradermal influenza vaccine elicits cross-reactive antibody responses against heterologous A(H3N2) influenza viruses.

The aim of the present study was to explore the ability of Intanza(®) 15 µg, the intradermal (ID) trivalent inactivated split-virion influenza vaccine containing 15 µg hemagglutinin per strain, to enhance the antibody responses against heterologous circulating H3N2 strains in adults 60 years and older. During the 2006-2007 influenza season, subjects aged 60 years or older were randomly assigned to receive one dose of ID or an intramuscular (IM, Vaxigrip(®)) influenza vaccine, which contained the reassortant A/Wisconsin/67/05(H3N2) strain as the H3N2 component. Antibody responses were assessed against the homologous vaccine strain, against the A/Brisbane/10/07(H3N2) reassortant strain and against four heterologous H3N2 field isolates (A/Genoa/62/05(H3N2), A/Genoa/3/07(H3N2), A/Genoa/2/07(H3N2), A/Genoa/3/06(H3N2)). The viruses tested belonged to three different clades that were closely related antigenically to A/California/7/04(H3N2), A/Nepal/921/06(H3N2) and A/Brisbane/10/07(H3N2). Antibody responses to these viruses were measured in 25 subjects per group using both haemagglutination inhibition (HI) and neutralization (NT) assays. At least one Committee for Medicinal Products for Human Use (CHMP) immunogenicity criteria for vaccine approval in the elderly was reached by both vaccines against all the viruses used in the study. All three CHMP criteria were reached against A/California/7/04(H3N2)-like, A/Nepal/921/06(H3N2)-like and A/Brisbane/10/07(H3N2)-like viruses by Intanza(®) 15 µg ID vaccine, while IM vaccination did not meet seroprotection criteria against circulating A/Nepal/921/06(H3N2)-like and A/Brisbane/10/07(H3N2)-like viruses or seroconversion criteria against A/Brisbane/10/07(H3N2)-like viruses. Post-vaccination HI titer, seroconversion, and seroprotection rates were higher against all viruses in subjects who received Intanza(®) 15 µg. The superiority of the seroprotection rate against the A/Nepal/921/06(H3N2)-like strain attained statistical significance despite the small sample size. Upon Beyer correction for pre-vaccination status, post-immunization HI titers against A/California/7/04(H3N2)-like and A/Brisbane/10/07(H3N2)-like strains and NT post-immunization titers against A/Wisconsin/67/05(H3N2), A/California/7/04(H3N2)-like, A/Brisbane/10/07(H3N2)-like strains were significantly higher in subjects immunized with Intanza(®) 15 µg than in individuals receiving IM vaccine. This study, although limited in the size of study population, demonstrated the broader immune response elicited by an ID influenza vaccine vs. a standard IM influenza vaccine against heterologous viruses including field isolates.

AF03-adjuvanted and non-adjuvanted pandemic influenza A (H1N1) 2009 vaccines induce strong antibody responses in seasonal influenza vaccine-primed and unprimed mice.

Pandemic influenza vaccines have been manufactured using the A/California/07/2009 (H1N1) strain as recommended by the World Health Organization. We evaluated in mice the immunogenicity of pandemic (H1N1) 2009 vaccine and the impact of prior vaccination against seasonal trivalent influenza vaccines (TIV) on antibody responses against pandemic (H1N1) 2009. In naïve mice, a single dose of unadjuvanted H1N1 vaccine (3 microg of HA) was shown to elicit hemagglutination inhibition (HI) antibody titers >40, a titer associated with protection in humans against seasonal influenza. A second vaccine dose of pandemic (H1N1) 2009 vaccine strongly increased these titers, which were consistently higher in mice previously primed with TIV than in naïve mice. At a low immunization dose (0.3 microg of HA), the AF03-adjuvanted vaccine elicited higher HI antibody titers than the corresponding unadjuvanted vaccines in both naïve and TIV-primed animals, suggesting a potential for antigen dose-sparing. These results are in accordance with the use in humans of a split-virion inactivated pandemic (H1N1) 2009 vaccine formulated with or without AF03 adjuvant to protect children and young adults against influenza A (H1N1) 2009 infection.

Emulsion-based adjuvants for influenza vaccines.

The ongoing epizootic of highly pathogenic avian H5N1 influenza and its direct transmissibility and high pathogenicity in humans has led to renewed interest in the development of influenza vaccines with enhanced immunogenicity. Influenza vaccines are currently under development against influenza strains that are potentially pandemic threats, such as H5N1, as well as against the current seasonal influenza strains for use in populations susceptible to severe influenza disease. Influenza vaccines may be generally divided into two types: seasonal vaccines for use in a population that is largely primed to subtypes of the circulating influenza A strains and pandemic influenza vaccines that are designed to protect against influenza A viruses of a hemagglutinin (HA) subtype, to which the vast majority of the population is immunologically naive. Pandemic influenza vaccines can be further subdivided into prepandemic vaccines produced for use prior to or just after the declaration of a pandemic, and pandemic influenza vaccines that would be produced and used only after a pandemic is declared. Prepandemic influenza vaccines are formulated using HA and neuraminidase, which are likely to be antigenically similar to the influenza virus subtype deemed to pose the most probable pandemic threat. Enhanced vaccine immunogenicity is desirable for pandemic influenza vaccines and for seasonal vaccines used in target populations, such as the elderly, in which vaccine responses against the circulating influenza subtypes may be weak. Various methods to enhance the immunogenicity of influenza vaccines are under evaluation. Along with dose escalation and alternative delivery routes, strategies for improving the immunogenicity of influenza vaccines have focused on the use of immunologic adjuvants. An adjuvanted seasonal influenza vaccine, Fluad, has been licensed in some countries in Europe since 1997 for the elderly population, and a number of clinical trials have been completed or are in progress evaluating the use of adjuvants with pandemic and seasonal influenza vaccines. This review will focus on the use of emulsion-based adjuvants for enhancing the immunogenicity of pandemic influenza vaccines and of seasonal influenza vaccines in target populations.

Heterosubtype neutralizing responses to influenza A (H5N1) viruses are mediated by antibodies to virus haemagglutinin.

BACKGROUND: It is increasingly clear that influenza A infection induces cross-subtype neutralizing antibodies that may potentially confer protection against zoonotic infections. It is unclear whether this is mediated by antibodies to the neuraminidase (NA) or haemagglutinin (HA). We use pseudoviral particles (H5pp) coated with H5 haemagglutinin but not N1 neuraminidase to address this question. In this study, we investigate whether cross-neutralizing antibodies in persons unexposed to H5N1 is reactive to the H5 haemagglutinin. METHODOLOGY/PRINCIPAL FINDINGS: We measured H5-neutralization antibody titers pre- and post-vaccination using the H5N1 micro-neutralization test (MN) and H5pp tests in subjects given seasonal vaccines and in selected sera from European elderly volunteers in a H5N1 vaccine trial who had detectable pre-vaccination H5N1 MN antibody titers. We found detectable (titer > or = 20) H5N1 neutralizing antibodies in a minority of pre-seasonal vaccine sera and evidence of a serological response to H5N1 in others after seasonal influenza vaccination. There was excellent correlation in the antibody titers between the H5N1 MN and H5pp tests. Similar correlations were found between MN and H5pp in the pre-vaccine sera from the cohort of H5N1 vaccine trial recipients. CONCLUSIONS/SIGNIFICANCE: Heterosubtype neutralizing antibody to H5N1 in healthy volunteers unexposed to H5N1 is mediated by cross-reaction to the H5 haemagglutinin.

A phase I clinical trial of a PER.C6 cell grown influenza H7 virus vaccine.

Avian influenza H7 viruses have transmitted from poultry to man causing human illness and fatality, highlighting the need for pandemic preparedness against this subtype. We have developed and tested the first cell-based human vaccine against H7 avian influenza virus in a phase I clinical trial. Sixty healthy volunteers were intramuscularly vaccinated with two doses of split H7N1 virus vaccine containing 12 microg or 24 microg haemagglutinin alone or with aluminium hydroxide adjuvant (300 microg or 600 microg, respectively). The vaccine was well tolerated in all subjects and no serious adverse events occurred. The vaccine elicited low haemagglutination inhibition and microneutralisation titres, although the addition of aluminium adjuvant augmented the antibody response. We found a higher number of antibody secreting cells and an association with IL-2 production in subjects with antibody response. In conclusion, our study shows that producing effective H7 pandemic vaccines is as challenging as has been observed for H5 vaccines.

A cell-based H7N1 split influenza virion vaccine confers protection in mouse and ferret challenge models.

BACKGROUND: In recent years, several avian influenza subtypes (H5, H7 and H9) have transmitted directly from birds to man, posing a pandemic threat. OBJECTIVES: We have investigated the immunogenicity and protective efficacy of a cell based candidate pandemic influenza H7 vaccine in pre-clinical animal models. METHODS: Mice and ferrets were immunised with two doses of the split virus vaccine (12-24 microg haemagglutinin) with or without aluminium hydroxide adjuvant and challenged 3 weeks after second dose with the highly pathogenic A/chicken/Italy/13474/99 (H7N1) virus. The H7N1-specific serum antibody response was also measured. After challenge, viral shedding, weight loss, disease signs and death (only mice) were recorded. RESULTS: Low-to-modest serum antibody titres were detected after vaccination. Nevertheless, the vaccine induced significant protection from disease after challenge with the wild-type virus. In the murine lethal challenge model, vaccination effectively prevented death and, furthermore, formulation with adjuvant reduced excessive weight loss and viral shedding. In ferrets, vaccination reduced viral shedding and protected against systemic spread of the virus. CONCLUSIONS: We have extended to the H7 subtype the finding that protective efficacy may not be directly correlated with the pre-challenge levels of serum antibodies, a finding which could be of great importance in assessing the potential effectiveness of pandemic influenza vaccines.

Measurement of neutralizing antibody responses against H5N1 clades in immunized mice and ferrets using pseudotypes expressing influenza hemagglutinin and neuraminidase.

Neutralizing antibody is associated with the prevention and clearance of influenza virus infection. Microneutralization (MN) and hemagglutination inhibition (HI) assays are currently used to evaluate neutralizing antibody responses against human and avian influenza viruses, including H5N1. The MN assay is somewhat labor intensive, while HI is a surrogate for neutralization. Moreover, use of replication competent viruses in these assays requires biosafety level 3 (BSL-3) containment. Therefore, a neutralization assay that does not require BSL-3 facilities would be advantageous. Toward this goal, we generated a panel of pseudotypes expressing influenza hemagglutinin (HA) and neuraminidase (NA) and developed a pseudotype-based neutralization (PN) assay. Here we demonstrate that HA/NA pseudotypes mimic release and entry of influenza virus and that the PN assay exhibits good specificity and reveals quantitative difference in neutralizing antibody titers against different H5N1 clades and subclades. Using immune ferret sera, we demonstrated excellent correlation between the PN, MN, and HI assays. Thus, we conclude that the PN assay is a sensitive and quantifiable method to measure neutralizing antibodies against diverse clades and subclades of H5N1 influenza virus.

Preparation of genetically engineered A/H5N1 and A/H7N1 pandemic vaccine viruses by reverse genetics in a mixture of Vero and chicken embryo cells.

BACKGROUND: In case of influenza pandemic, a robust, easy and clean technique to prepare reassortants would be necessary. OBJECTIVES: Using reverse genetics, we prepared two vaccine reassortants (A/H5N1 x PR8 and A/H7N1 x PR8) exhibiting the envelope glycoproteins from non-pathogenic avian viruses, A/Turkey/Wisconsin/68 (A/H5N9) and A/Rhea/New Caledonia/39482/93 (A/H7N1) and the internal proteins of the attenuated human virus A/Puerto Rico/8/34 (H1N1). METHODS: The transfection was accomplished using a mixture of Vero and chicken embryo cells both of which are currently being used for vaccine manufacturing. RESULTS: This process was reproducible, resulting in consistent recovery of influenza viruses in 6 days. Because it is mainly the A/H5N1 strain that has recently crossed the human barrier, it is the A/PR8 x A/H5N1 reassortant (RG5) that was further amplified, either in embryonated hen eggs or Vero cells, to produce vaccine pre-master seed stocks that met quality control specifications. Safety testing in chickens and ferrets was performed to assess the non-virulence of the reassortant, and finally analysis using chicken and ferret sera immunized with the RG5 virus showed that the vaccine candidate elicited an antibody response cross-reactive with the Hong Kong 1997 and 2003 H5N1 strains but not the Vietnam/2004 viruses. CONCLUSIONS: The seeds obtained could be used as part of a pandemic vaccine strain 'library' available in case of propagation in humans of a new highly pathogenic avian strain.