Characterization of an influenza A H5N2 reassortant as a candidate for live-attenuated and inactivated vaccines against highly pathogenic H5N1 viruses with pandemic potential☆
Introduction
Repeated outbreaks of H5N1 influenza in Asia continue to pose a pandemic threat to human health. Highly pathogenic avian influenza (HPAI) A (H5N1) viruses were first recognized to cause respiratory disease in humans in 1997 when viruses from infected poultry were transmitted to humans, causing 18 documented cases including six fatalities [1], [2], [3]. In 2003, H5N1 virus reemerged in humans to infect two family members in Hong Kong resulting in the death of one person [4]. Since late 2003, unprecedented numbers of HPAI H5N1 outbreaks in poultry have occurred in many Asian, European and African countries, resulting in more than 220 laboratory-confirmed human cases with a fatality rate of greater than 50% [5]. Thus, the development of safe, dose-sparing and effective human vaccines against H5N1 influenza is a high priority for global public health.
Since 1997, HPAI H5N1 viruses from birds have undergone rapid genetic evolution [6], [7], [8]. The viruses isolated from humans have reflected this genetic variation with concomitant antigenic variation. H5N1 viruses from 2004 to 2005 comprise two genetically distinct virus clades, both of which are antigenically distinct from the 2003 human isolates, which in turn were antigenically distinct from those isolated from humans in 1997 [9], [10], [11]. Once recognized to cause human disease, new candidate vaccine strains must be generated for each H5N1 antigenic variant.
A number of different strategies have been applied to generate vaccine candidates against HPAI H5N1 viruses, including the use of antigenically related non-pathogenic viruses to produce an inactivated influenza vaccine (IIV) and the use of purified recombinant HA protein. Both of these approaches have been evaluated clinically with suboptimal results [12], [13], [14]. More recently reverse genetics techniques have been optimized to allow for the generation of vaccine reassortant strains that possess HA with the modified multibasic cleavage site, which is associated with virulence in birds, and internal genes derived from a human vaccine donor strain [15], [16], [17]. This approach allows for the inclusion of an HA protein, albeit modified, that is antigenically highly related to that found in the circulating HPAI H5N1 virus.
Development of live-attenuated influenza vaccine (LAIV) for pandemic preparedness has certain advantages over other vaccine strategies. Since LAIV may provide effective protection against a broader range of variants, an exact match between the vaccine strain and circulating viruses may be less critical. As an example, LAIV was shown to provide highly effective protection in healthy pre-school children against a drift variant of influenza A (H3N2) in a pre-licensure study of LAIV in the USA [18]. Similar data were obtained in Russia (reviewed in [19]). The heterotypic efficacy of LAIV may be, at least in part, due to the induction of enhanced IgA antibody responses in the respiratory tract compared with those induced by IIV [20], [21]. Furthermore, since vaccine will be in short supply during a pandemic, multiple vaccine production options may be important.
Here we evaluate an H5 pandemic vaccine candidate created using classical reassortment techniques from an antigenically related non-pathogenic avian influenza H5N2 and an influenza cold-adapted (ca) donor strain A/Leningrad134/17/57 (H2N2; Len17) [22] for its protective efficacy against antigenically heterologous HPAI H5N1 strains. The H5 pandemic vaccine candidate (Len17/H5) possesses the HA from non-pathogenic A/Duck/Potsdam/1402-6/86 (H5N2; Pot/86) virus and all other genes from Len17 (7:1 genome composition) [23]. Len17/H5 demonstrated ca and ts phenotypes in vitro similar to those of the Len17 ca donor strain, grew to high titers in embryonated eggs and shared antigenic similarity with the H5N1 viruses isolated from humans in 1997 [23]. We demonstrate that the reassortant Len17/H5 virus is attenuated in mice and non-infectious for chickens, and effectively protects mice against heterologous HPAI H5N1 infection when used as either an LAIV or IIV. These results suggest a pandemic vaccine strategy that does not require reverse genetics technology, a heightened bio-safety level, or a precise antigenic match for vaccine strain generation, yet may offer protection against a heterologous virus in the early phase of a pandemic.
Section snippets
Viruses
The high-growth reassortant Len17/H5 that contained the HA gene from the non-pathogenic A/Duck/Potsdam/1402-6/86 (H5N2) virus (Pot/86) and other genes from the ca attenuated A/Leningrad/134/17/57 (H2H2) strain (Len17) was obtained in accordance with Ghendon et al, [24]. Wildtype H5N1 viruses used in this study were A/Hong Kong/156/97 (HK/156), A/Hong Kong/483/97 (HK/483), and A/Hong Kong/213/03 (HK/213). Viruses were propagated in the allantoic cavity of 10-day-old embryonated hens’ eggs at 34
Pathotyping and replication of Len17/H5 vaccine in chickens
The two parent and reassortant Len17/H5 viruses were administered to specific pathogen free (SPF) chickens to determine their potential risk for animal agriculture which included assessment of the ability to cause morbidity and mortality following i.v. inoculation (pathogenicity) and the level of tissue-specific replication following simulated natural exposure (i.n. inoculation). With i.v. or i.n. inoculation, no clinical disease signs or deaths were observed in the chickens with any of the
Discussion
A vaccine that is a close antigenic match with the circulating pandemic strain is optimal for the control of pandemic influenza, but such a vaccine may not be available for at least 6 months after the identification of a pandemic strain. In the interim, a vaccine that is an imperfect antigenic match may still be useful in protecting from severe disease or death. In this proof of concept study, we evaluated the immunogenicity and efficacy of a 7:1 reassortant H5 LAIV candidate generated from a
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The findings and conclusion in this report are those of the authors and do not necessarily represent the views of the funding agency, the US Department of Health and Human Services.
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These authors contributed equally to this study.