Generation of DelNS1 Influenza Viruses: a Strategy for Optimizing Live Attenuated Influenza Vaccines

Pui Wang, Min Zheng, Siu-Ying Lau, Pin Chen, Bobo Wing-Yee Mok, Siwen Liu, Honglian Liu, Xiaofeng Huang, Conor J Cremin, Wenjun Song, Yixin Chen, Yik-Chun Wong, Haode Huang, Kelvin Kai-Wong To, Zhiwei Chen, Ningshao Xia, Kwok-Yung Yuen, Honglin Chen, Pui Wang, Min Zheng, Siu-Ying Lau, Pin Chen, Bobo Wing-Yee Mok, Siwen Liu, Honglian Liu, Xiaofeng Huang, Conor J Cremin, Wenjun Song, Yixin Chen, Yik-Chun Wong, Haode Huang, Kelvin Kai-Wong To, Zhiwei Chen, Ningshao Xia, Kwok-Yung Yuen, Honglin Chen

Abstract

Nonstructural protein 1 (NS1) of influenza virus is a key virulence element with multifunctional roles in virus replication and a potent antagonist of host immune response. Deletion of NS1 (DelNS1) would create a safer and more extensively immunogenic live attenuated influenza virus (LAIV) vaccine. However, DelNS1 viruses are very difficult to grow in regular vaccine-producing systems, which has hampered the application of DelNS1 LAIV vaccines in humans. We have developed two master backbones of deleted-NS1 (DelNS1) viral genomes from influenza A or B viruses which contain novel adaptive mutations to support DelNS1-LAIV replication. These DelNS1-LAIVs are highly attenuated in human cells in vitro and nonpathogenic in mice but replicate well in vaccine-producing cells. Both influenza A and influenza B DelNS1 LAIVs grow better at 33°C than at 37 to 39°C. Vaccination with DelNS1 LAIV performed once is enough to provide potent protection against lethal challenge with homologous virus and strong long-lasting cross protection against heterosubtypic or antigenically distantly related influenza viruses in mice. Mechanistic investigations revealed that DelNS1-LAIVs induce cross protective neutralizing antibody and CD8+ and CD4+ T cell immunities. Importantly, it has been shown that DelNS1-LAIV can be used to enhance specific anti-influenza immunity through expression of additional antigens from the deleted-NS1 site. Generation of DelNS1 viruses which are nonpathogenic and able to grow in vaccine-producing systems is an important strategy for making highly immunogenic LAIV vaccines that induce broad cross protective immunity against seasonal and emerging influenza.IMPORTANCE Current seasonal influenza vaccines are suboptimal and low in immunogenicity and do not provide long-lasting immunity and cross protection against influenza virus strains that have antigenically drifted. More-effective influenza vaccines which can induce both humoral immunity and T cell immunity are needed. The NS1 protein of influenza virus is a virulence element and the critical factor for regulation of the host immune response during virus infection. Deletion of the NS1 protein is a strategy to make an optimal LAIV vaccine. However, DelNS1 viruses are very difficult to grow in regular vaccine-producing systems, hampering the application of DelNS1 LAIV vaccines in humans. We have generated a panel of both influenza A and influenza B DelNS1 LAIVs which are able to grow in regular vaccine-producing cells. These DelNS1 LAIV vaccines are completely nonpathogenic, exhibit potent and long-lasting immunity, and can be used to express extra viral antigen to induce cross protective immunity against seasonal and emerging influenza.

Keywords: NS1; influenza vaccines; live attenuated vaccine.

Copyright © 2019 Wang et al.

Figures

FIG 1
FIG 1
Generation and characterization of CA4-DelNS1 virus. (A) Full-length and NS1 gene-deleted NS segments. NCR, noncoding region. (B) Confirmation of NS1 deletion in rescued CA4-DelNS1 virus by RT-PCR and agarose gel electrophoresis. -ve, negative control. (C) Newly rescued CA4-DelNS1 virus was passaged in MDCK cells 20 times to promote growth adaptation. After the virus titers stabilized, the entire genome of the CA4-DelNS1 virus was analyzed and adaptive mutations were identified. (D) Effect of adaptive mutations on the efficiency of CA4-DelNS1 virus rescue. DelNS1 pHW2000 plasmids with individual or combined adaptive mutations or the original DelNS1 plasmid (DelNS1-WT) were transfected together with plasmids encoding the other 7 viral segments into 293T/MDCK cell mixtures and incubated at 33°C or 37°C. After 72 h, viral supernatants were collected and titrated. (E) Growth of CA4-DelNS1 at 33°C or 37°C was analyzed. MDCK cells or embryonated chicken eggs were infected with CA4-DelNS1 or WT virus at 0.1 MOI or 1,000 PFU, respectively. Viral supernatants or allantoic fluids were collected at the indicated time points and titrated by plaque assay in MDCK cells. Data represent mean values ± standard deviations of results from three independent experiments. Statistical comparisons between means were performed by Student's t test. ***, P < 0.001; **, P < 0.01; *, P < 0.05; NS, not significant.
FIG 2
FIG 2
Avirulence of CA4-DelNS1 virus. (A) A549 cells were infected with CA4-WT virus, with ca-LAIV, or with CA4-DelNS1 virus at 0.01 MOI and cultured at 33°C or 37°C. ca-LAIV is a cold-adapted 2009 H1N1 virus that was isolated from the licensed LAIV vaccine (17). Viral supernatants were collected at different time points and titrated by plaque assay in MDCK cells. Data represent mean values ± standard deviations of results from three independent experiments. Statistical comparisons between means were performed by Student's t test. ***, P < 0.001; **, P < 0.01. (B) DelNS1 virus infection in mice. Groups of six 6-week-old mice were inoculated with 106 PFU of CA4-DelNS1, H7N9-DelNS1, WSN-DelNS1, or H7N9 WT virus. PBS was used as an inoculation control. Body weight and survival were observed for 14 days. Data represent mean body weight values ± standard deviations of results from 6 mice.
FIG 3
FIG 3
CA4-DelNS1 LAIV vaccination protects mice from both homologous and heterologous virus challenge. (A) Timelines for vaccination and virus challenge. Mice were intranasally vaccinated once with CA4-DelNS1 virus (2 × 106 PFU), ca-LAIV (2 × 106 PFU), or PBS and then challenged with virus after 3 weeks. (B to D) Vaccinated mice were challenged with 10 MLD50 CA4 (H1N1) mouse-adapted virus (B), 10 MLD50 H7N9 virus (C), or 100 MLD50 H5N1 virus (D). Body weights and survival were observed for 14 days. Body weight data represent mean values ± standard deviations of results from 6 mice. (E) HI analysis of sera from mice vaccinated with CA4-DelNS1, ca-LAIV, or PBS, collected prior to virus challenge.
FIG 4
FIG 4
CA4-DelNS1 as a master backbone for construction of DelNS1 H3N2 LAIV. (A) Generation of CA4-DelNS1-HK4801 (H3N2) by plasmid transfection. CA4-DelNS1-HK4801 (H3N2) virus was rescued with HA and NA plasmids derived from the A/Hong Kong/4801/2014 (H3N2) strain and 6 internal gene plasmids from the CA4-DelNS1 virus containing adaptive mutations, as described for Fig. 1. (B) CA4-DelNS1-HK4801 cannot replicate in IFN-competent A549 cells. (C) CA4-DelNS1-HK4801 replication efficiency varied with temperature in MDCK cells. (D) CA4-DelNS1-HK68 (H3N2) protects mice from challenge with homologous HK68-MA virus. Mice were vaccinated with CA4-DelNS1-HK68 (HK68-WT) virus (2 × 106 PFU) or PBS and after 3 weeks were challenged with HK68-MA (mouse-adapted) virus. (E) CA4-DelNS1-HK4801 protects mice from challenge with heterologous HK68-MA virus. Mice were vaccinated with CA4-DelNS1-HK4801 (H3N2) virus (3 × 107 PFU) or PBS and were challenged later with HK68-MA virus. Body weight and survival were observed for 14 days. Body weight data represent mean values ± standard deviations of results from 6/7 mice. Statistical comparisons between means were performed by Student's t test. ***, P < 0.001; **, P < 0.01; *, P < 0.05.
FIG 5
FIG 5
Generation and characterization of DelNS1 B LAIV. (A) Generation of DelNS1-B8038 influenza B virus by reverse genetics. pHW2000 plasmids containing an NS1-deleted (DelNS1) segment and the seven remaining genome segments derived from B/Hong Kong/8038/2011 (Victoria) virus were transfected into a 293T/MDCK cell mixture. Rescued virus was passaged in MDCK cells until the virus titer stabilized. (B) Adaptive mutations in growth-adapted DelNS1-B8038 influenza B virus. Mutations in PA (T210C), NA (T1429C), NP (C182T), and M (G88C plus A280G) were identified. (C) Growth of DelNS1-B8038 influenza B virus in cell culture. MDCK or A549 cells were infected with either WT or DelNS1-B8038 virus at 0.01 MOI and incubated at 33°C or 37°C for 72 h. Virus supernatants were collected and titrated by plaque assay in MDCK cells. Data represent mean values ± standard deviations of results from three independent experiments. Statistical comparisons between means were performed by Student's t test. ***, P < 0.001. (D) DelNS1-B8038 (B-DelNS1) LAIV vaccination protected mice from challenge with both Victoria and Yamagata lineage mouse-adapted viruses. Mice were vaccinated with DelNS1-B8038 (2 × 106 PFU) or PBS. After 3 weeks, vaccinated mice were challenged with mouse-adapted B/Brisbane/60/2008 (Victoria) or B/Florida/4/2006 (Yamagata) influenza B virus at 10 MLD50. Body weight and survival were observed for 14 days. Body weight data represent mean values ± standard deviations of results from 6 mice.
FIG 6
FIG 6
DelNS1 LAIV induces cross protective T cell responses against influenza viruses. (A) Induction of antigen-specific CD8+ and CD4+ T cells by CA4-DelNS1 LAIV was estimated by intracellular cytokine staining (ICS) of ex vivo peptide-stimulated splenocytes, followed by flow cytometric analysis. (B) Schedule of T cell depletion and virus challenge. Vaccinated mice were injected intraperitoneally (i.p.) with 100 μg of anti-CD8α or anti-CD4 or with both anti-CD8α and anti-CD4 or with isotype control (IgG2b) antibodies on day 17 (D17), D19, and D21 after immunization and day 3 after virus challenge. (C) Mice were challenged with H7N9 virus (10 MLD50) on day 21 after vaccination and monitored for 14 days. (D) Mice were challenged with mouse-adapted CA4 (H1N1) virus (10 MLD50) and monitored for 14 days. Body weight data represent mean values ± standard deviations of results from 7 mice. Statistical comparisons between means were performed by Student's t test. ***, P < 0.001; **, P < 0.01.
FIG 7
FIG 7
DelNS1 LAIV expression of B-HA1 antigen protected against lethal challenge with both influenza A and B viruses in mice. (A) Illustration of cloning of B-HAI region into the CA4-DelNS1 NS segment. (B) Mice were vaccinated once with CA4-DelNS1 and then challenged 3 weeks later with mouse-adapted H1N1 influenza A virus (A/California/04/2009, 10 MLD50) or influenza B virus (B/Brisbane/60/2008, 10 MLD50) and monitored for 14 days. (C) Mice were vaccinated twice, 1 month apart, with 106 PFU of either CA4-DelNS1 virus or CA4-DelNS1-BHA1, which contains the HA1 gene of influenza B virus (B/Brisbane/60/2008) at the site in the NS segment from which NS1 was deleted. Mice were challenged with mouse-adapted B/Brisbane/60/2008 (10 MLD50) 1 month after the second vaccination and monitored for 2 weeks. (D) Mice were vaccinated once with 1 × 107 PFU of either CA4-DelNS1-HK4801 (H3N2) virus or CA4-DelNS1-HK4801-BHA1 LAIV. Vaccinated mice were challenged with mouse-adapted B/Brisbane/60/2008 (10 MLD50) 1 month after the second dose of vaccination. Mice were monitored for body weight change and survival for 2 weeks. Body weight data represent mean values ± standard deviations of results from 6 (B and C) or 7 (D) mice.

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