The Immunogenicity of a Cell‐derived H7N1 Split Influenza Virion Vaccine in Mice (2009)

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 μg HA) formulated with or without aluminium hydroxide adjuvant (60 μg Al). Sera were collected at various time points after vaccination and the H7N1-specific 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 μg 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 μg 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 μg group and the lowest response found in mice vaccinated with 0.2 μg 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 μg 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. The serum antibody response induced after vaccination with a cell-derived H7N1 vaccine. (A) The haemagglutinin inhibition (HI) titres were measured in a modified HI assay using cell grown RD3 H7N1 virus and horse red blood cells at 3 weeks after the first (n = 6, triangle) and second (n = 10, diamond) vaccination. Each symbol represents one animal from the adjuvanted (+, filled symbols) or non-adjuvanted (−, open symbols) vaccine or control (C, circles) groups and the line indicates the geometric mean titre (GMT). (B) Beads coated with cell-grown whole RD3 virus were used to measure the kinetics of the serum IgG response in a bead-based immunoassay at 3 weeks after the first (n = 6) and second (n = 10) vaccination. Filled bars represent aluminium-adjuvanted vaccine groups (+) whereas open bars represent the non-adjuvanted vaccine groups (−). Mice were vaccinated with 0.2, 3, 6 or 12 μg HA as indicated. Data are presented as the mean fluorescent intensity ± standard error of the mean. 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 [14], showing that the vaccine formulation itself influenced the IgG2a/IgG1 profile and may be due to the inherent immunogenicity of the viral ssRNA. In the present study we have shown that only low antibody titres are elicited to a cell-based inactivated split virion H7N1. However, this vaccine effectively prevented systemic spread, reduced viral shedding and reduced illness and/or death after challenge of mice and ferrets with the highly pathogenic parent avian H7 virus [1, 2]. Others have earlier found that the protective efficacy of candidate pandemic H5N1 vaccines does not necessarily correlate with pre-challenge serum antibody levels [7, 15-17], a finding which could be of great importance in assessing the potential effectiveness of pandemic influenza vaccines. Therefore, a pandemic vaccine that does not fulfil the current licensing criteria, used for licensing seasonal influenza vaccines, for antibody induction may still offer considerable protection. There is thus a clear need for efficient adjuvants and further investigation of the role of other immunological factors in protection, such as the cytokine profile or effective priming via stimulation of the innate and cellular immune responses. There is currently widespread concern about the low serum antibody responses observed in humans after vaccination with pandemic avian virus candidates and clearly further animal studies and ultimately human clinical trials are needed to elucidate the reasons for the low antibody responses and to define how this correlates with protective efficacy. This study was funded by the European Union (Flupan QLK2-CT-2001-01786) and we thank all of the partners involved in the Flupan project. We also thank Nina Harkestad and the staff at the animal houses at the University of Bergen for their help and care of the mice.