Frederick R. Vogel

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.

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.

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

Abstract 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.

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 ≥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 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.

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.

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.

The Immunogenicity of a Cell-derived H7N1 Split Influenza Virion Vaccine in Mice

To the Editor: In the last decade a number of avian influenza subtypes (H5, H7, H9) have crossed the species barrier into man causing illness and death, reminding the world of the pandemic potential of influenza. Although the H5N1 virus is responsible for the majority of avian influenza zoonoses worldwide, in Europe and North America, human cases of avian influenza have almost exclusively been caused by the H7 subtype. Therefore, the WHO prioritizes both avian H5 and H7 for pandemic vaccine development. During an influenza pandemic the timely and widespread use of an appropriately formulated influenza pandemic vaccine will be a vital prophylactic measure. The current worldwide manufacturing capacity based on embryonated hens’ eggs is not expected to meet the global pandemic vaccine demand and cell culture systems provide an attractive alternative, allowing for a more flexible and rapid scale-up of influenza vaccine production. In man, two doses of adjuvanted candidate pandemic inactivated vaccine are generally necessary to induce assumed protective serum antibody levels. Currently, aluminium salts are the most commonly used adjuvant in licensed vaccines. In this study we have investigated the immunogenicity of a candidate pandemic cell-derived influenza H7 vaccine alone or formulated with aluminium hydroxide adjuvant in a murine model. The vaccine strain (RD3) was generated by reverse genetics, using the neuraminidase and modified haemagglutinin (HA) from the highly pathogenic A ⁄ chicken ⁄ Italy ⁄ 13474 ⁄ 99 (H7N1) strain, and subsequently propagated in PER.C6 cells. Mice were subcutaneously immunized with two doses of the split virus vaccine (0.2–12 lg HA) formulated with or without aluminium hydroxide adjuvant (60 lg Al). Sera were collected at various time points after vaccination and the H7N1specific serum antibody response measured by modified haemagglutination inhibition, microneutralization, luminescent bead based and ELISA assays. Considerable numbers of animal and human pandemic H5N1 vaccination studies have been conducted; however, this is the first candidate pandemic H7 vaccine to undergo preclinical protective efficacy studies [1, 2] and complete a Phase I clinical trial [3]. In this study, we found that two doses of 12 lg HA of a cell-based inactivated split virion H7N1 vaccine are required to elicit low HI antibody titres in mice (Fig. 1A). Furthermore, formulation of the vaccine with aluminium hydroxide adjuvant enhanced the immunogenicity of 12 lg HA of this vaccine (all mice responded with HI titres ranging from 8 to 128) and neutralizing antibody was detected in 5 of 10 mice with titres ranging from 20 to 133. After the first vaccination, very low levels of IgG antibodies were detected in all groups by the luminescent bead immunoassay (Fig. 1B). The second vaccination significantly (P < 0.05, t-test) boosted the response in all groups, with the highest antibody level found in the adjuvanted 12 lg group and the lowest response found in mice vaccinated with 0.2 lg vaccine. The levels of antibodies detected for each group appeared to plateau from 7 days after the second dose. Formulation of the vaccine with aluminium hydroxide adjuvant significantly (P < 0.05, t-test) enhanced the antibody response after both vaccinations in the group immunized with 12 lg HA and also increased the antibody response in the other vaccine groups. Our results confirm and extend previous animal studies [4–7] showing that development of H7 pandemic vaccines will be similarly challenging to H5N1 vaccines and that an effective adjuvant is also needed for cell grown H7 pandemic vaccines. Activation of the innate immune system through recognition of the natural pathogen-associated molecular pattern in the vaccine probably influences its immunogenicity. Recently it has been found that ssRNA of influenza is present in whole virus vaccines and stimulates toll-like receptor (TLR) 7 and leads to a T helper (Th)1 profile [8]. In our study we further assessed the quality of the T helper response to this pandemic split virus vaccine by using the IgG1 as a marker of a Th2 response and IgG2a antibody response as an indicator of a Th1-biased response [9]. IgG1 antibodies have been found to be associated with virus neutralization, whilst IgG2a is correlated with clearance of virus [10]. In both the aluminium-adjuvanted and non-adjuvanted vaccine groups the IgG response consisted almost exclusively of IgG1, with significantly lower levels of IgG2a detected (P < 0.05, t-test) suggesting a Th2-biased response (data not shown) [2]. We have previously observed a mixed Th1 ⁄ Th2 profile after vaccination with egg-grown seasonal split influenza virus vaccines in mice [11–13], whereas formulation of vaccines with aluminium hydroxide adjuvant is known to skew the response to Th2 in mice. Interestingly, in mice vaccinated with whole H7N1 vaccine or infected with the highly pathogenic parent virus a mixed IgG2a ⁄ IgG1 profile was observed L E T T E R T O T H E E D I T O R doi: 10.1111/j.1365-3083.2009.02254.x ..................................................................................................................................................................