The development of a novel vaccine against avian influenza H7N9 virus
Influenza A viruses (IAV) are classified into the Orthomyxoviridae family and are composed of segmented, negative-sense, single-stranded ribonucleic acid (ssRNA) genomes in an enveloped particle. IAV is capable of infecting a wide variety of species, including but not limited to humans, birds, pigs, bats, and sea mammals. Up until 2013, H7N9 IAV was only prevalent in poultry; however, at this time H7N9 began infecting humans in China. Since 2013, China has seen six epidemic waves of the H7N9 virus, with human cases and deaths totaling 1,568 and 616, respectively, as of March 4, 2020. Although this virus presents high morbidity and mortality rates in humans, the majority of human cases have been a result of close contact to poultry in live poultry markets (LPMs). Fortunately, the change for sustained human-to-human transmission has not yet been acquired in this virus. However, due to IAVs evolutionary mechanisms of antigenic shift and antigenic drift, H7N9 could gain sustained transmission among humans at any time, which poses a severe threat to public health. Therefore, preventive control methods must be developed in an effort to control the spread of this influenza virus. Vaccination is currently the main method for controlling the spread and preventing influenza infection. Currently, the two available types of vaccines are the inactivated influenza vaccines (IIV) and the live attenuated influenza vaccines (LAIV). IIVs are composed of either whole influenza virions or portions of its virion produced in large volumes, followed by inactivation of the virus with either β-propiolactone or formaldehyde. IIVs are commonly administered intramuscularly, and sometimes include adjuvants to boost their immune responses. IIVs have been countlessly demonstrated to be highly safe for all populations. LAIVs on the other hand, are composed of live viruses of which their virulence is reduced through limited replication in the vaccinated host. LAIVs are administered intranasally and do not require an adjuvant because they are capable of stimulating stronger immune responses compared to IIVs. However, one common drawback towards the use of LAIVs is the possibility of virulence reversion. On the contrary, replication-defective virus vaccines are made up of viruses defective in either viral replication, synthesis, or assembly. These replication-defective virus vaccines, therefore, consist of very limited replication in the vaccinated host and have been found to possess the advantages of both IIVs and LAIVs. These advantages include the high safety profile due to the low risk of virulence reversion, as well as the ability to induce a strong immune response. To date, no commercial vaccine is available for the H7N9 influenza viruses. The first step in the influenza replication cycle is the binding of the virus to the host cell, which is followed by receptor-mediated endocytosis. After endocytosis occurs, in order for influenza virus to become infective, cleavage of the hemagglutinin (HA) precursor form, HA0, into HA1 and HA2 must occur. This cleavage is most often mediated by trypsin-like host proteases, inducing fusion between the viral and endosomal membranes. Therefore, this particular step is essential for determining viral pathogenicity. For this Masters project, the goal was to generate a replication-defective virus vaccine derived from H7N9 IAV, that is composed of an altered HA cleavage site that can only be cleaved and thus activated in vitro by the exogenous protease elastase which is not readily available in the respiratory tract. This replication-defective virus vaccine would, therefore, be inactive during natural infection, but active in vitro if the appropriate protease was provided. Previous studies have proven this replication-defective nature through the mutation of the HA cleavage site from a trypsin-sensitive motif to an elastase-sensitive motif. However, these studies have only been performed with the swine influenza virus (SIV) H1N1, a human-derived H7N7 HPAI, a mouse-adapted human-derived H1N1 virus, as well as an influenza B virus (IBV) (Babiuk et al., 2011; Gabriel et al., 2008; Mamerow et al., 2019; Masic et al., 2009; Masic et al., 2010; Masic et al., 2013; Stech et al., 2011; Stech et al., 2005). Using the technique of reverse genetics, we generated a recombinant mutant H7N9 virus, BC15-HA/QTV/NA (PR8), derived from the human isolate A/British Columbia/01/2015 (H7N9) [BC15 (H7N9)] with a backbone from A/Puerto Rico/8 (H1N1) [PR8 (H1N1)]. This recombinant mutant BC15-HA/QTV/NA (PR8) virus possesses a mutant HA composed of three mutations at the HA cleavage site: lysine to glutamine at amino acid (aa) 337 (Lys-Gln), glycine to threonine at aa 338 (Gly-Thr), and arginine to valine at aa 339 (Arg-Val). In addition to the mutant HA, this recombinant mutant BC15-HA/QTV/NA (PR8) virus also contains the neuraminidase (NA) from BC15 (H7N9) and the six internal proteins from PR8 (H1N1). In the first part of our study, we established a mouse model of BC15 (H7N9) influenza virus. BALB/c mice were intranasally infected with various doses of BC15 (H7N9) (103 PFU, 104 PFU, and 105 PFU), and were monitored daily for 14 days post-infection (d.p.i.). In this study, we found BC15 (H7N9) to affect mice in a dose-dependent manner: the 103 dose killing all mice by 8 d.p.i.; the 104 dose by 6 d.p.i.; and the 105 dose by 5 d.p.i. In addition, all doses were capable of inducing high viral replication, pathology, and proinflammatory cytokine induction in the mouse lung. From this study, we concluded 103 PFU to be the chosen dose for future experiments. In the second part of this study, we developed and characterized the recombinant mutant BC15-HA/QTV/NA (PR8) virus, which showed this virus to be entirely dependent on elastase for its replication, contain similar growth properties to its wild-type counterpart, and be genetically stable in vitro. In addition, when this recombinant mutant BC15-HA/QTV/NA (PR8) virus was intranasally administered in BALB/c mice, it was found to be non-virulent and replication-defective, evident by a lack of body weight loss, 100% survival rate, and no viral replication detected in the mouse lung. Since we established this recombinant mutant BC15-HA/QTV/NA (PR8) virus to be replication-defective in mice, in order to consider this virus as a replication-defective virus vaccine candidate, we needed to test the immunogenicity and protective efficacy in BALB/c mice. To do this, we intranasally vaccinated mice twice with this recombinant mutant BC15-HA/QTV/NA (PR8) virus and then challenged the mice with a lethal dose of BC15 (H7N9). In this study, we reported that the intranasally administered BC15-HA/QTV/NA (PR8) virus induced significantly elevated levels of antigen-specific IFN-γ and IL-5 secreting cells in the splenocytes, which is evidence of a strong cell-mediated response. In addition, this virus increased the levels of neutralizing antibodies in the mouse serum, evident by both the hemagglutinin inhibition (HAI) and serum virus neutralization (SVN) assays, as well as heightened the levels of antigen-specific IgG, IgG1, and IgG2a in the mouse serum. Once the mice were challenged with BC15 (H7N9), our data showed that two intranasal vaccinations with BC15-HA/QTV/NA (PR8) were sufficient to provide complete protection of the mice from a homologous challenge. This complete protection was evident by the lack of body weight loss, 100% survival rate, lack of viral replication detected in the mouse lung, as well as the complete abolishment of proinflammatory cytokine induction in the mouse lung associated with the influenza disease. Taken together, this study demonstrates the strong potential the BC15-HA/QTV/NA (PR8) virus possesses to serve as a replication-defective virus vaccine candidate against H7N9 influenza viruses.
Influenza A virus H7N9, recombinant mutant H7N9 virus, replication-defective virus vaccine
Master of Science (M.Sc.)
School of Public Health
Vaccinology and Immunotherapeutics