Elsevier

Vaccine

Volume 31, Issue 33, 18 July 2013, Pages 3339-3346
Vaccine

Immunogenicity and protective efficacy of cold-adapted X-31 live attenuated pre-pandemic H5N1 influenza vaccines

https://doi.org/10.1016/j.vaccine.2013.05.080Get rights and content

Highlights

  • Using reverse genetics, we generated live attenuated H5N1 influenza vaccines.

  • The vaccines showed desired level of safety both in vitro and in vivo.

  • The vaccines were highly immunogenic in mouse model.

  • The vaccines elicited cross-clade antibody responses to heterologous H5N1 strains.

  • The vaccines provided solid protection against heterologous H5N2 infection in mice.

Abstract

Despite global efforts to control influenza viruses, they have taken a heavy toll on human public health worldwide. Among particular threats is highly pathogenic avian H5N1 influenza virus (HPAI) due to not only its high mortality in humans but also possible human-to-human transmission either through reassortment with other human influenza viruses such as 2009 pandemic H1N1 influenza virus, or by genetic mutations. With the aim of developing effective vaccines against the H5N1 viruses, we generated two live attenuated H5N1 vaccine candidates against A/Indonesia/05/2005 (clade 2.1) and A/chicken/Korea/ES/2003 (clade 2.5) strains, in the genetic background of the cold-adapted donor strain of X-31. In mice, a single dose of immunization with each of the two vaccines was highly immunogenic inducing high titers of serum viral-neutralizing and hemagglutinin-inhibiting antibodies against the homologous H5N1 strain. Furthermore, significant levels of cross-clade antibody responses were induced by the vaccines, suggesting a broad-spectrum cross-reactivity against the heterologous H5N1 strains. The immunizations provided solid protections against heterologous lethal challenges with H5N2 virus, significantly reducing the morbidity and challenge virus replications in the respiratory tracts. The robustness of the antibody responses against both the homologous and heterologous strains, together with efficient protection against the lethal H5N2 challenge, strongly support the protection against wild type H5N1 infections. These results could serve as an experimental basis for the development of safe and effective H5N1 pre-pandemic vaccines while further addressing the biosecurity concerns associated with H5N1 HPAI.

Introduction

Since the first report of human infections in 1997, the highly pathogenic avian influenza (HPAI) H5N1 virus has continued to spread among avian species and occasionally transmitted to humans, resulting in high case-fatality rates. Furthermore, the sudden emergence and global circulation of the 2009 pandemic H1N1 virus (pdmH1N1) by direct human-to-human transmission caused a concern for potential genetic exchange between HPAI H5N1 and the pdmH1N1, which would give rise to reassortants with both high virulence and transmissibility among humans [1], [2].

Such persistent and increasing threats posed by the HPAI emphasize the need of effective vaccines. Several inactivated H5N1 vaccines elicited protective humoral immune responses in animals or humans in multiple doses with adjutants [3], [4], [5], [6]. Baculovirus-expressed recombinant protein vaccine and subvirion vaccine were also tested for their immunogenicity but were poorly immunogenic, requiring at least two doses and the use of adjuvant [7], [8]. Live attenuated influenza vaccine (LAIV) is becoming a more attractive alternative because of several distinctive advantages, including stimulation of both systemic and mucosal immunity, ease of intranasal administration, induction of cell-mediated immunity, and cross-reactivity against antigenically distinct strains [9], [10], [11]. To date, three different cold-adapted influenza A (H2N2) viruses have been developed as the donor strains of LAIVs; A/Ann Arbor/6/60 ca (H2N2), A/Leningrad/134/17/57 ca (H2N2), and A/Leningrad/134/47/57 ca (H2N2) [12], [13], [14]. These cold-adapted strains not only have served as general platforms for generating LAIVs against seasonal influenza viruses, but also for pre-pandemic vaccines against avian H5N1 or H5N2 virus, with robust immunogenicity and protective efficacy in various clinical and preclinical trials [15], [16], [17], [18], [19].

Previously, we generated a cold-adapted influenza A virus, X-31 ca, which demonstrated excellent profiles of productivity, safety, immunogenicity, and protective efficacy in the mouse model [20]. The vaccine also provided extremely early protection against heterologous and heterosubtypic challenges, mediated by an antibody-independent innate immune response [21]. We also established a reverse genetics platform to generate this X-31 ca virus from cloned cDNAs derived from influenza viral RNAs [22], making it possible to generate 6:2 reassortant vaccine candidates through a simple DNA transfection protocol [23]. Using the reverse genetic system with X-31 ca we recently generated live attenuated influenza vaccine against the 2009 pandemic H1N1 virus and evaluated its immunogenicity and protective efficacy against homologous and heterologous infections in animal models [24], [25]. Here we extended the vaccine study to H5N1 virus and evaluated immunogenicity and protective efficacy using a mouse model. We generated two different live attenuated H5N1 pandemic vaccines candidates, each carrying HA and NA from A/Indonesia/05/2005 (H5N1) or A/chicken/Korea/ES/2003 (H5N1). These two H5N1 vaccine candidates were evaluated for their attenuated phenotypes, immunogenicity against both homologous and heterologous H5N1 strains, and protection against lethal heterosubtypic challenges with H5N2 virus.

Section snippets

Generation of H5N1 ca vaccines

The HA and NA of two reassortant H5N1 ca vaccines, R-IN2005 and R-CK2003, were derived from A/Indonesia/05/2005 (clade 2.1) and A/Chicken/Korea/ES/2003 (clade 2.5), respectively. Another reassortant H5N1 virus, R-VN2004, consisting of HA and NA derived from A/Vietnam/1203/2004 (clade 1) was also generated with the same backbone for the analysis of cross-reactivity. Each HA and NA gene was synthesized and cloned into pHW2000 vector via reverse genetics as previously described [23]. To ensure the

Generation and growth properties of H5N1 ca vaccines

Using a reverse genetics system, we generated two 6:2 reassortant viruses, R-IN2005 and R-CK2003, containing the HA and NA genes from the A/Indonesia/05/2005 (clade 2.1) or A/Chicken/Korea/ES/2003 (clade 2.5), in the genetic background of the cold-adapted X-31 (X-31 ca) [20], [22]. To determine whether these two viruses maintain the cold-adapted (ca) and temperature-sensitive (ts) phenotypes, their growth properties at various temperatures in cell cultures and embryonated chicken eggs were

Discussion

The X-31 ca-based H5N1 vaccines exhibited ca and ts phenotypes in eggs demonstrating both robust growth at lower temperatures and restrictive growth at higher temperatures. Based on the previous results that the X-31 ca backbone manifested such phenotypes in its growth ability in eggs [20], it is reasonable to conclude that a limited growth of the H5N1 ca vaccines at higher temperature is afforded by the six internal genes from the backbone strain. However, both vaccines exhibited slightly

Role of the funding source

This work was supported by the R&D Programs of Korean governments: MKE [grant number: 10031969], MEST [grant number: 2010-0001932], MHW [grant number: A085105], and Korea CDC [grant number: 2009-E00522-00]. This work was also supported from Yonsei University Research Fund 2012.

Conflicts of interest

The authors declare no conflicts of financial interests.

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    1

    These authors contributed equally to this article.

    2

    Present address: CJ Pharmaceutics, South Korea.

    3

    Present address: Department of Biotechnology, The Catholic University, South Korea.

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