Qiliang Liu

Mutations of two transmembrane cysteines of hemagglutinin (HA) from influenza A H3N2 virus affect HA thermal stability and fusion activity

Influenza A H3N2 virus caused 1968 Hong Kong influenza pandemic, and has since been one of the most prevalent seasonal influenza viruses in global populations, representing a credible pandemic candidate in future. Previous studies have established that the hemagglutinin (HA) protein is the predominant antigen and executes receptor binding and membrane fusion. Homologous sequence analysis of all HA subtypes of influenza viruses revealed that two cysteine residues (540 and 544) are uniquely present in the transmembrane domain (TM) of HA proteins from all influenza A H3N2 viruses. However, the functions of these two cysteines have not been fully studied. Here, we generated three mutants (C540S, C544L, and 2C/SL) to investigate the effects of the two TM cysteines on the biological functions of H3 HA. We herein presented evidences that the mutations of one or two of the cysteines did not affect the proper expressions of HA proteins in cells, and more importantly all mutant H3 HAs showed decreased thermal stability but increased fusion activity in comparison with wildtype HA. Our results taken together demonstrated that the two TM cysteines are important for the biological functions of H3 HA proteins.

Recombinant influenza H1, H5 and H9 hemagglutinins containing replaced H3 hemagglutinin transmembrane domain showed enhanced heterosubtypic protection in mice

Abstract Influenza A viruses cause annual epidemics and irregular pandemics. A vaccine with heterosubtypic protection (hetero-protection) has been needed. In the present study, various influenza H1, H3, H5, and H9 hemagglutinin (HA) proteins were expressed in insect cells, and then mice were subcutaneously immunized with the expressed HA proteins, and challenged by influenza A viruses (A/Puerto Rico/8/1934 (H1N1) or A/chicken/Guangdong/96 (H9N2)). The results first showed that wild-type H3 hemagglutinin (HA) (H3-WT), but not a transmembrane domain (TM) mutant, had hetero-protection against both H1N1 and H9N2 with survival rates of 17% and 33% respectively, and that wild-type H1 (H1-WT), H5 (H5-WT) and H9 (H9-WT) had no hetero-protection against H1N1 or H9N2 except for H5-WT against H1N1 with a survival rate of 17%. Then the H3-WT TM replaced the TMs of H1-WT, H5-WT and H9-WT to generate recombinant H1-TM, H5-TM and H9-TM respectively, and whether the H3-WT TM-dependent hetero-protection could be transferred to these TM mutants was investigated. The results showed that the H3-WT TM-dependent hetero-protection was transferable. H1-TM against H9N2 and H9-TM against H1N1 were with survival rates of 33% and 17% respectively, and H5-TM against both H1N1 and H9N2 with survival rates of 50% and 17% respectively. Furthermore, higher dosage H5-TM scored 100% hetero-protection against H1N1. These results demonstrated that replacement of the TMs of non-H3 HAs with H3-WT TM could enhance their hetero-protection. These findings would help the development of future influenza vaccines against pandemics such as the recently appeared H7N9 infection.

Production and immunogenicity of chimeric virus-like particles containing porcine reproductive and respiratory syndrome virus GP5 protein.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a severe threat in swine industry and causes heavy economic losses worldwide. Currently, the available vaccines are the inactivated and attenuated virus vaccines, but the use of PRRSV in their production raises the issue of safety. We developed a chimeric virus-like particles (VLPs) vaccine candidate for PRRSV protection. The chimeric VLPs was composed of M1 protein from H1N1 influenza virus and a fusion protein, denoted as NA/GP5, containing the cytoplasmic and transmembrane domains of H1N1 virus NA protein and PRRSV GP5 protein. Vaccination of BALB/c mice with 10 μg of chimeirc VLPs by intramuscular immunization stimulated antibody responses to GP5 protein, and induced cellular immune response. The data suggested that the chimeric VLP vaccine candidate may provide a new strategy for further development of vaccines against PRRSV infection.

Assembly and immunological properties of a bivalent virus-like particle (VLP) for avian influenza and Newcastle disease

Avian influenza virus (AIV) and Newcastle disease virus (NDV) are both important pathogens in poultry worldwide. The protection of poultry from avian influenza and Newcastle disease can be achieved through vaccination. We embarked on the development of a bivalent vaccine that would allow for a single immunization against both avian influenza and Newcastle disease. We constructed a chimeric virus-like particle (VLP) that is composed of the M1 protein and HA protein of avian influenza virus and a chimeric protein containing the cytoplasmic and transmembrane domains of AIV neuraminidase protein (NA) and the ectodomain of the NDV hemagglutinin-neuraminidase (HN) protein (NA/HN). The single immunization of chickens with the chimeric VLP vaccine induced both AIV H5- and NDV-specific antibodies. The HI titers and specific antibodies elicited by the chimeric VLPs were statistically similar to those elicited in animals vaccinated with the corresponding commercial monovalent vaccines. Chickens vaccinated with chimeric VLP vaccine and then challenged with the Newcastle disease F48E9 virus displayed complete protection. Overall, the chimeric VLP vaccine elicits strong immunity and can protect against Newcastle disease virus challenge.

Evidences for the existence of intermolecular disulfide-bonded oligomers in the H3 hemagglutinins expressed in insect cells

The hemagglutinin (HA) protein as the predominant antigen, executes receptor binding and membrane fusion, which critically influence the virological characteristics of influenza viruses. The literature contained scattered data showing reduction-sensitive HA oligomers when HA proteins were analyzed under non-reducing conditions. However, whether the reduction-sensitive HA oligomers are inter-monomer disulfide-bonded has not been studied. Here, we showed: (1) the detection of β-mercaptoethanol-sensitive H3 HA oligomers was not affected by the treatment of cells with iodoacetamide prior to cell solubilization; (2) H3 HA oligomers were present on cell surfaces; (3) H3 HA oligomers had higher density than monomers; and (4) mutation of all the five C-terminal cysteines completely abolished the formation of H3 HA oligomers. Furthermore, mutant HAs with mutations of TM cysteines, CT cysteines or all five cysteines had decreased thermal stability but increased fusion activity in comparison with wildtype HA. In conclusion, this study has presented enough evidence for the existence of inter-monomer S–S H3 HA oligomers formed by five C-terminal cysteines, and suggested that all five C-terminal cysteines exerted opposite effects on HA thermal stability and fusion activity.

Recombinant influenza A H3N2 viruses with mutations of HA transmembrane cysteines exhibited altered virological characteristics.

Influenza A H3N2 virus as the cause of 1968 pandemic has since been circulating in human and swine. Our earlier study has shown that mutations of one or two cysteines in the transmembrane domain of H3 hemagglutinin (HA) affected the thermal stability and fusion activity of recombinant HA proteins. Here, we report the successful generation of three recombinant H3N2 mutant viruses (C540S, C544L, and 2C/SL) with mutations of one or two transmembrane cysteines of HA in the background of A/swine/Guangdong/01/98 [H3N2] using reverse genetics, indicating that the mutated cysteines were not essential for virus assembly and growth. Further characterization revealed that recombinant H3N2 mutant viruses exhibited larger plaque sizes, increased growth rate in cells, enhanced fusion activity, reduced thermal and acidic resistances, and increased virulence in embryonated eggs. These results demonstrated that the transmembrane cysteines (C540 and C544) in H3 HA have profound effects on the virological features of H3N2 viruses.

Production and immunogenicity of chimeric virus-like particles containing the spike glycoprotein of infectious bronchitis virus.

Infectious bronchitis virus (IBV) poses a severe threat to the poultry industry and causes heavy economic losses worldwide. Vaccination is the most effective method of preventing infection and controlling the spread of IBV, but currently available inactivated and attenuated virus vaccines have some disadvantages. We developed a chimeric virus-like particle (VLP)-based candidate vaccine for IBV protection. The chimeric VLP was composed of matrix 1 protein from avian influenza H5N1 virus and a fusion protein neuraminidase (NA)/spike 1 (S1) that was generated by fusing IBV S1 protein to the cytoplasmic and transmembrane domains of NA protein of avian influenza H5N1 virus. The chimeric VLPs elicited significantly higher S1-specific antibody responses in intramuscularly immunized mice and chickens than inactivated IBV viruses. Furthermore, the chimeric VLPs induced significantly higher neutralization antibody levels than inactivated H120 virus in SPF chickens. Finally, the chimeric VLPs induced significantly higher IL-4 production in mice. These results demonstrate that chimeric VLPs have the potential for use in vaccines against IBV infection.

Chimeric influenza-virus-like particles containing the porcine reproductive and respiratory syndrome virus GP5 protein and the influenza virus HA and M1 proteins

Both porcine reproductive and respiratory syndrome and swine influenza are acute, highly contagious swine diseases. These diseases pose severe threats for the swine industry and cause heavy economic losses worldwide. In this study, we have developed a chimeric virus-like particle (VLP) vaccine candidate for porcine reproductive and respiratory syndrome virus (PRRSV) and H3N2 influenza virus and investigated its immunogenicity in mice. The HA and M1 proteins from the H3N2 influenza virus and the PRRSV GP5 protein fused to the cytoplasmic and transmembrane domains of the NA protein were both incorporated into the chimeric VLPs. Analysis of the immune responses showed that the chimeric VLPs elicited serum antibodies specific for both PRRSV GP5 and the H3N2 HA protein, and they stimulated cellular immune responses compared to the responses to equivalent amounts of inactivated viruses. Taken together, the results suggested that the chimeric VLP vaccine represents a potential strategy for the development of a safe and effective vaccine to control PRRSV and H3N2 influenza virus.

Influenza bivalent vaccine comprising recombinant H3 hemagglutinin (HA) and H1 HA containing replaced H3 hemagglutinin transmembrane domain exhibited improved heterosubtypic protection immunity in mice.

Influenza caused by infection of influenza viruses is still a leading cause of morbidity and mortality in human. Vaccination is the main defense against influenza virus, but current influenza trivalent or quatrivalent vaccines (TIV/QIV) would lose their effectiveness when vaccine strains are mismatched with circulating strains. Our early study showed that recombinant influenza Hx-TM HA proteins containing H3 HA transmembrane domain(TM) had improved immunogenicity and heterosubtypic protection over corresponding wild-type Hx-WT HA proteins. In present study, bivalent vaccines containing H3-WT+Hx-TM were investigated for their immune responses and heterosubtypic protection immunities. The data showed that the bivalent vaccines containing H3-WT and H5-TM or H1-TM had improved immune responses and heterosubtypic protection over the bivalent vaccines containing H3-WT and H5-WT or H1-WT respectively. These results demonstrated that the improved immune responses and heterosubtypic protection of Hx-TM HA proteins could be translated into bivalent vaccines, suggesting a feasible strategy of improving the immune responses and heterosubtypic protection of influenza multivalent vaccines such as TIV and QIV.

Recombinant influenza H9N2 virus with a substitution of H3 hemagglutinin transmembrane domain showed enhanced immunogenicity in mice and chicken

In recent years, avian influenza virus H9N2 undergoing antigenic drift represents a threat to poultry farming as well as public health. Current vaccines are restricted to inactivated vaccine strains and their related variants. In this study, a recombinant H9N2 (H9N2-TM) strain with a replaced H3 hemagglutinin (HA) transmembrane (TM) domain was generated. Virus assembly and viral protein composition were not affected by the transmembrane domain replacement. Further, the recombinant TM-replaced H9N2-TM virus could provide better inter-clade protection in both mice and chickens against H9N2, suggesting that the H3-TM-replacement could be considered as a strategy to develop efficient subtype-specific H9N2 influenza vaccines.