Elsevier

Antiviral Research

Volume 143, July 2017, Pages 97-105
Antiviral Research

A recombinant H7N9 influenza vaccine with the H7 hemagglutinin transmembrane domain replaced by the H3 domain induces increased cross-reactive antibodies and improved interclade protection in mice

https://doi.org/10.1016/j.antiviral.2017.03.029Get rights and content

Highlights

  • The recombinant H7N9-53TM virus with the H7 hemagglutinin transmembrane domain (TM) replaced by the H3 TM was rescued.

  • Inactivated H7N9-53TM vaccine induces increased cross-reactive antibodies and improved interclade protection.

  • The H3 TM domain replacement technology might be applicable for other subtypes of influenza viruses.

Abstract

Influenza A H7N9 virus is the latest emerging pandemic threat, and has rapidly diverged into three clades, demanding a H7N9 virus vaccine with broadened protection against unmatched strains. Hemagglutinin (HA)-based structural design approaches for stabilizing HA proteins have provided excitingly promising results. However, none of the HA-based structural design approaches has been applied to a recombinant replicative influenza virus. Here we report that our HA-based structural design approach is a first in the field to generate a recombinant replicative H7N9 virus (H7N9-53TM) showing broadened protection. The H7N9-53TM contains a replaced H3 HA transmembrane domain (TM) in its HA protein. In mice, the inactivated H7N9-53TM vaccine induced significantly higher HI titers, HA-specific IgG titers, and IFN-γ production than the corresponding H7N9-53WT inactivated virus vaccine containing wild-type HA. More excitingly, mice immunized with the H7N9-53TM showed full protection against homologous (H7N9-53) and interclade (H7N9-MCX) challenges with minimal weight loss, no detectable lung viral loads, and no apparent pulmonary lesions and inflammation, while mice immunized with the H7N9-53WT showed partial protection (only 60% against H7N9-MCX) with severe weight loss, detectable lung viral loads, and severe pulmonary lesions and inflammation. In summary, this study presents a better vaccine candidate (H7N9-53TM) against H7N9 pandemics. Furthermore, our HA-based structural design approach would be conceivably applicable to other subtype influenza viruses, especially the viruses from emerging pandemic and epidemic influenza viruses such as H5N1 and H1N1.

Introduction

Avian influenza H7N9 virus is the latest looming pandemic threat when it made its first leap in China from chickens to humans in February 2013 (Bao et al., 2013, Gao et al., 2013). Until November 2016, 800 cases of infection, with a case fatality rate of approximately 35–40%, have been reported by the World Health Organization (WHO) (Organization, 2016). There is no sustained H7N9 virus transmission among humans yet. However, the facts that H7N9 is able to transmit via direct contact in ferret and guinea pig models and novel strain infections occurred in blood-related family members suggest the possibility of the H7N9 transmission in humans (Belser et al., 2013, Liu et al., 2014b, Zhu et al., 2013). Moreover, since H7 subtype influenza viruses do not currently circulate in humans, humans are immunologically naive to this subtype. Therefore, H7N9 presents grave public health concerns worldwide.

Vaccines have been the main defense against influenza since the inactivated influenza virus vaccine was introduced more than 70 years ago (Francis, 1953). The most widely used seasonal influenza vaccines are the trivalent/quadrivalent inactivated vaccines (TIV/QIV) (Wong and Webby, 2013). However, current influenza vaccines do not possess the desired broadened immunity and protection. The development of an effective vaccine against H7N9 has been a challenge; for instance, Wong et al. reported that in ferrets a single dose of the inactivated H7N9 vaccine cannot prevent virus replication in lung completely (Wong et al., 2014). In addition, H7N9 viruses like other influenza viruses diverge into distinct genotypes (clades), making a sufficiently effective broad-spectrum vaccine an urgent need (Watanabe et al., 2014).

Hemagglutinin (HA) is a homotrimeric molecule that is the main surface glycoprotein and major immunogenic protein of influenza virus. To increase the cross-reactive immunity of influenza vaccines, previous studies have employed various HA-based structural design approaches to stabilize HA proteins by fusing a trimerization sequence such as bacteriophage T4 fibritin foldon (Wei et al., 2008), GCN4PII trimerization (Weldon et al., 2010), and ferritin (Kanekiyo et al., 2013) to the HA extracellular domain. However, these approaches suffer in at least two practical aspects: one is that the trimerization sequences are alien to HA proteins, raising safety concerns for being used as vaccine in humans; the other is that these HA fusion proteins cannot be incorporated into a replicative influenza virus so that they are not compatible with current influenza vaccine manufacturing technologies and facilities.

Our group has developed a new HA-based structural design approach that replaces the transmembrane domains (TM) of non-H3 HA proteins with the TM of H3 HA protein to stabilize trimeric HA proteins. Our earlier studies replaced the TM of H1, H5 and H9 HA proteins with the H3 HA TM and the results demonstrated that these modified trimeric H1, H5 and H9 HA proteins expressed in insect cells increase their thermal stability and cross-reactive immunity and protection over their wild-type counterparts (Liu et al., 2014a, Liu et al., 2015, Xu et al., 2013). In this study, we intended to generate a better H7N9 vaccine by investigating whether a recombinant H7N9 virus containing a H7 HA protein with a replaced H3 HA TM could be rescued and further whether the inactivated vaccine prepared by the rescued H7N9 virus could provide better protection against the challenges of homologous and interclade H7N9 strains in mice. Our results showed that such a recombinant H7N9 virus was successfully rescued, and that the rescued recombinant H7N9 virus vaccine elicited significantly increased immune responses and cross-reactive protection.

Section snippets

Cell lines

Human embryonic kidney cells (293T) and Madin-Darby Canine Kidney (MDCK) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Thermo Scientific), penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37 °C with 5% CO2. Insect cells (Sf9) were maintained as suspension cultures in serum free Sf900II medium (Gibco) at 27 ± 2 °C.

Expression and purification of seven H7 HA proteins

The HA segments from the following seven H7N9 strains were synthesized:

A/Chicken/Guangdong/53/2014(H7N9)

Generation and characterization of recombinant H7N9-53WT and H7N9-53TM

To investigate the impact of the replacement of the H7 HA TM with a H3 HA TM on its compatibility in replicative viruses, we attempted to rescue in parallel two recombinant H7N9 viruses, H7N9-53WT and H7N9-53TM (identical to H7N9-53WT except for a replaced H3 HA TM in the H7 HA) (Fig. 1A). Both the H7N9-53WT and H7N9-53TM were successfully rescued, showing similarly typical influenza virus morphologies under electron microscopy (Fig. 1B), and comparable protein compositions under SDS-PAGE (

Discussion

This study is a first in the field to improve the protective immunity of a replicative influenza virus through our HA-based structural design approach that replaces the H7 HA TM with a H3 HA TM. The resultant recombinant H7N9-53TM virus is a superior inactivated vaccine over the recombinant H7N9-53WT virus in all aspects investigated in this study, including higher HI titers, higher HA-specific IgG titers, higher HA-specific IFN-γ production, lower weight loss, higher survival rate, lower lung

Conflict of interest statement

GDL has patent applications pertinent to this study. All others declare no conflict of interest.

Acknowledgements

This work was supported by Guangzhou Science and Technology Plan (201504010025), Science and Technology Plan of Guangdong (2013B020224003), Guangdong Natural Science Foundation (2015A030313095) and H7N9 Avian Influenza Joint Research (2014-1046).

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