Elsevier

The Lancet

Volume 357, Issue 9272, 16 June 2001, Pages 1937-1943
The Lancet

Early Report
Safety and antigenicity of non-adjuvanted and MF59-adjuvanted influenza A/Duck/Singapore/97 (H5N3) vaccine: a randomised trial of two potential vaccines against H5N1 influenza

https://doi.org/10.1016/S0140-6736(00)05066-2Get rights and content

Summary

Background

In 1997, pathogenic avian influenza A/Hong Kong/97 (H5N1) viruses emerged as a pandemic threat to human beings. A non-pathogenic variant, influenza A/Duck/Singapore/97 (H5N3), was identified as a leading vaccine candidate. We did an observer-blind, phase I, randomised trial in healthy volunteers to assess safety, tolerability, and antigenicity of MF59-adjuvanted and non-adjuvanted vaccines.

Methods

32 participants were randomly assigned MF59, and 33 non-adjuvanted vaccine. Two doses were given 3 weeks apart, of 7·5, 15, or 30 μg haemagglutinin surface-antigen influenza A H5N3 vaccine. Antibody responses were measured by haemagglutination inhibition, micro-neutralisation, and single radial haemolysis (SRH). The primary outcome was geometric mean antibody titre 21 days after vaccination.

Findings

The A/Duck/SIngapore vaccines were safe and well tolerated. Antibody response to non-adjuvanted vaccine was poor, the best response occurring after two 30 μg doses: one, four, four, and one person of eleven seroconverted by haemagglutination inhibition, microneutralisation, H5N3 SRH, and H5N1 SRH, respectively. The geometric mean titres of antibody, and seroconversion rates, were significantly higher after MF59 adjuvanted vaccine. Two 7·5 μg doses of MF59 adjuvanted vaccine gave the highest seroconversion rates: haemagglutination inhibition, six of ten; microneutralisation, eight often; H5N3 SRH, ten often; H5N1 SRH, nine of ten. Geometric mean titre of antibody to the pathogenic virus, A/Hong Kong/489/97 (H5N1), was about half that to A/Duck/Singapore virus.

Interpretation

Non-adjuvanted A/Duck/Singapore/97 (H5N3) vaccines are poorly immunogenic and doses of 7·5–30 μg haemagglutinin alone are unlikely to give protection from A/Hong Kong/97 (H5N1) virus. Addition of MF59 to A/Duck/Singapore/97 vaccines boost the antibody response to protection levels. Our findings have implications for development and assessment of vaccines for future pandemics.

Introduction

The natural hosts and reservoirs of all 15 haemagglutinin and all nine neuraminidase subtypes of influenza A are free-living aquatic birds. Pandemics of influenza in man occur unpredictably as a result of so-called antigenic shift. In the past century, pandemics emerged in 1918, 1957, and 1968. Although the genetic origins of the 1918 pandemic virus are uncertain, the pandemic strains of 1957 and 1968 were human-avian reassortants.1

Until recently, avian influenza viruses were thought to be not transmissable directly to human beings. People were not susceptible to an H5 virus infection that caused high poultry mortality in Pennsylvania in 1983;2 although this conclusion is based on the haemagglutinin-inhibition test, which might underestimate human serological response to avian inflenza viruses. Experimental infection of human beings with H4, H6, and H10 subtypes of avian influenza resulted in limited viral replication, minimal symptoms, and no detectable haemagglutination-inhibition antibody response.3 However, H7 avian viruses have been associated with two cases of conjunctivitis in human beings.4, 5 Any doubt that influenza A viruses can cross naturally into people and cause severe disease was dispelled in 1997, when avian H5N1 influenza caused six human deaths and 18 hospital admissions in Hong Kong.6

The H5N1 infections in people were preceded by the circulation of highly pathogenic H5N1 viruses in Hong Kong poultry.6, 7 All genes of the human H5N1 viruses were avian-like, but the viruses replicated systemically in mice, were neurotropic, and caused lethal disease without the adaptation usually required of human influenza A viruses.8 H5N1 infection of human beings caused severe morbidity and longlasting admission to hospital.9 Although Katz and colleagues'10 serological results suggested that H5N1 virus was not efficiently transmitted from person-to-person, laboratory and clinical findings indicate that a human pandemic could be caused by an avian virus.

One strategy to develop vaccines against pathogenic avian influenza viruses is to use antigenically related strains that are non-pathogenic to birds and presumably non-pathogenic to man. With this approach, vaccines and reagents can be prepared safely with equipment and facilities that are used to develop and prepare traditional interpandemic vaccines and reagents. Preliminary studies with ferret antisera showed that isolates of H5N1 virus from the 18 human cases in the Hong Kong outbreak could be divided into two closely related groups: A/Hong Kong/156/97 (HK/156, group A), and A/Hong Kong/483/97 (HK483, group B).

In December, 1997, the UK National Institute for Biological Standards and Control prepared several possible vaccine strains against H5N1. A non-pathogenic avian virus, A/Duck/Singapore-Q/F1 19–3/97 (H5N3), was antigenically similar enough to human and avian A/HongKong/97 (H5N1) isolates to be tested as a vaccine. Two 10 or 15 μg doses of vaccine (inactivated whole virus) prepared from this virus protected mice from lethal challenge with influenza HK/483 or HK156.8, 11 Two doses were needed to elicit a haemagglutination-inhibition response. This response was substantially increased by aluminium hydroxide adjuvant. However, this H5N3 virus did not grow well in hens' eggs, and thus was not suitable for vaccine production. Attempts were made to improve growth by recombination with influenza virus A/Puerto Rico/8/34 (H1N1, PR8). The resulting virus, NIB-40, was antigenically the same as A/Duck/Singapore/97, but the virus yield grew by 30%. However, no PR8 genes were detected in NIB-40, so it was probably not a recombinant. Wood and colleagues11 showed that vaccines prepared from NIB-40 protected mice from a lethal dose of influenza A/HongKong/97 (H5N1). Martin12 showed that MF59 adjuvant increases the antibody responses of man to surface-antigen influenza vaccines.

We undertook a phase I, single-centre, two-dose trial of non-adjuvanted, monovalent surface-antigen A/Duck/Singapore/97 vaccine and the same vaccine adjuvanted with MF59. We aimed to: measure humoral immune responses and short-term local and systemic reactions to the vaccines, assess A/Duck/Singapore/97 virus as a vaccine candidate, and get an idea of the amount of vaccine needed to raise protective levels of antibody in human beings.

Section snippets

Participants

The study was done during May-July, 1999, in the Leicester Royal Infirmary, UK. Eligible participants were healthy volunteers aged 18–40 years. Exclusion criteria were: anaphylaxis; allergy to eggs, antibiotics, mercury-containing products, or vaccine; immunosuppression as a result of disease or treatment; treatment with immuno-stimulants; pregnancy or refusal of women to use a reliable method of contraception during the study; receipt of blood products or immunoglobulins in the preceding 3

Participants

We included 65 volunteers (figure 1, table 1), most of whom were nurses; hence there was a greater number of women than men in the study. Baseline characteristics did not differ between treatment groups. One volunteer was withdrawn from the study on day 21 because of tonsillitis. Before vaccination, all participants had serum H5 haemagglutination-inhibition titres of less than 1 in 10, and neutralising antibody titres of less than 1 in 20. No prevaccination serum samples reacted by SRH.

Adverse events

Both

Discussion

Although well tolerated, all doses of non-adjuvanted surface-antigen vaccine that were prepared from influenza A/Duck/Singapore/97 virus were poorly immunogenic, which suggests that this vaccine would provide little protection against influenza A/Hong Kong/97.

The haemagglutination-inhibition test was substantially less sensitive than microneutralisation and SRH. However, each assay showed that MF59-adjuvanted surface antigen vaccine gave significantly higher geometric mean titres of H5N3 and

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