Glycosylation generates an efficacious and immunogenic vaccine against H7N9 influenza virus.

Zoonotic avian influenza viruses pose severe health threats to humans. Of several viral subtypes reported, the low pathogenic avian influenza H7N9 virus has since February 2013 caused more than 1,500 cases of human infection with an almost 40% case-fatality rate. Vaccination of poultry appears to reduce human infections. However, the emergence of highly pathogenic strains has increased concerns about H7N9 pandemics. To develop an efficacious H7N9 human vaccine, we designed vaccine viruses by changing the patterns of N-linked glycosylation (NLG) on the viral hemagglutinin (HA) protein based on evolutionary patterns of H7 HA NLG changes. Notably, a virus in which 2 NLG modifications were added to HA showed higher growth rates in cell culture and elicited more cross-reactive antibodies than did other vaccine viruses with no change in the viral antigenicity. Developed into an inactivated vaccine formulation, the vaccine virus with 2 HA NLG additions exhibited much better protective efficacy against lethal viral challenge in mice than did a vaccine candidate with wild-type (WT) HA by reducing viral replication in the lungs. In a ferret model, the 2 NLG-added vaccine viruses also induced hemagglutination-inhibiting antibodies and significantly suppressed viral replication in the upper and lower respiratory tracts compared with the WT HA vaccines. In a mode of action study, the HA NLG modification appeared to increase HA protein contents incorporated into viral particles, which would be successfully translated to improve vaccine efficacy. These results suggest the strong potential of HA NLG modifications in designing avian influenza vaccines.

A Single Amino Acid in the Polymerase Acidic Protein Determines the Pathogenicity of Influenza B Viruses.

Influenza B virus (IBV) is one of the human respiratory viruses and one of the targets of seasonal vaccination. However, the bifurcation of two antigenically distinct lineages of IBVs makes it difficult to arrange proper medical countermeasures. Moreover, compared with pathogenicity-related molecular markers known for influenza A virus, little has been known for IBVs. To understand pathogenicity caused by IBVs, we investigated the molecular determinants of IBV pathogenicity in animal models. After serial lung-to-lung passages of Victoria lineage B/Brisbane/60/2008 (Vc_BR60) and Yamagata lineage B/Wisconsin/01/2010 (Ym_WI01) viruses in BALB/c mice, we identified the mouse-adapted Vc_BR60 (maVc_BR60) and Ym_WI01 (maYm_WI01) viruses, respectively. To find a molecular clue(s) to the increased pathogenicity of maVc_BR60 and maYm_WI01, we determined their genetic sequences. Several amino acid mutations were identified in the PB2, PB1, PA, BM2, and/or NS1 protein-coding regions, and one concurrent lysine (K)-to-arginine (R) mutation in PA residue 338 (PA K338R) was found in both maVc_BR60 and maYm_WI01 viruses. When analyzed using viruses rescued through reverse genetics, it was shown that PA K338R alone could increase the pathogenicity of both IBVs in mice and viral replication in the respiratory tracts of ferrets. In a subsequent minireplicon assay, the effect of PA K338R was highlighted by the enhancement of viral polymerase complex activity of both Vc_BR60 and Ym_WI01 viruses. These results suggest that the PA K338R mutation may be a molecular determinant of IBV pathogenicity via modulating the viral polymerase function of IBVs.IMPORTANCE To investigate molecular pathogenic determinants of IBVs, which are one of the targets of seasonal influenza vaccines, we adapted both Victoria and Yamagata lineage IBVs independently in mice. The recovered mouse-adapted viruses exhibited increased virulence, and of the various mutations identified from both mouse-adapted viruses, a concurrent amino acid mutation was found in the PA protein-coding region. When analyzed using viruses rescued through reverse genetics, the PA mutation alone appeared to contribute to viral pathogenicity in mice within the compatible genetic constellation between the IBV lineages and to the replication of IBVs in ferrets. Regarding the potential mechanism of increased viral pathogenicity, it was shown that the PA mutation could upregulate the viral polymerase complex activity of both IBV lineages. These results indicate that the PA mutation could be a newly defined molecular pathogenic determinant of IBVs that substantiates our understanding of the viral pathogenicity and public health risks of IBVs.

Distinct molecular evolution of influenza H3N2 strains in the 2016/17 season and its implications for vaccine effectiveness.

Influenza virus is a respiratory pathogen that causes seasonal epidemics by resulting in a considerable number of influenza-like illness (ILI) patients. During the 2016/17 season, ILI rates increased unusually earlier and higher than previous seasons in Korea, and most viral isolates were subtyped as H3N2 strains. Notably, the hemagglutinin (HA) of most Korean H3N2 strains retained newly introduced lysine signatures in HA antigenic sites A and D, compared with that of clade 3C.2a vaccine virus, which affected antigenic distances to the standard vaccine antisera in a hemagglutination inhibition assay. The neuraminidase (NA) of Korean H3N2 strains also harbored amino acid mutations. However, neither consistent amino acid mutations nor common phylogenetic clustering patterns were observed. These suggest that Korean H3N2 strains of the 2016/17 season might be distantly related with the vaccine virus both in genotypic and phenotypic classifications, which would adversely affect vaccine effectiveness.

Effects of HA and NA glycosylation pattern changes on the transmission of avian influenza A(H7N9) virus in guinea pigs.

Avian influenza H7N9 virus has posed a concern of potential human-to-human transmission by resulting in seasonal virus-like human infection cases. To address the issue of sustained human infection with the H7N9 virus, here we investigated the effects of hemagglutinin (HA) and neuraminidase (NA) N-linked glycosylation (NLG) patterns on influenza virus transmission in a guinea pig model. Based on the NLG signatures identified in the HA and NA genetic sequences of H7N9 viruses, we generated NLG mutant viruses using either HA or NA gene of a H7N9 virus, A/Anhui/01/2013, by reverse genetics on the 2009 pandemic H1N1 virus backbone. For the H7 HA NLG mutant viruses, NLG pattern changes appeared to reduce viral transmissibility in guinea pigs. Intriguingly, however, the NLG changes in the N9 NA protein, such as a removal from residue 42 or 66 or an addition at residue 266, increased transmissibility of the mutant viruses by more than 33%, 50%, and 16%, respectively, compared with a parental N9 virus. Given the effects of HA-NA NLG changes with regard to viral transmission, we then generated the HA-NA NLG mutant viruses harboring the H7 HA of double NLG addition and the N9 NA of various NLG patterns. As seen in the HA NLG mutants above, the double NLG-added H7 HA decreased viral transmissibility. However, when the NA NLG changes occurred by a removal of residue 66 and an addition at 266 were additionally accompanied, the HA-NA NLG mutant virus recovered the transmissibility of its parental virus. These demonstrate the effects of specific HA-NA NLG changes on the H7N9 virus transmission by highlighting the importance of a HA-NA functional balance.

Effects of heat-killed Lactobacillus plantarum against influenza viruses in mice.

The potential use of dietary measures to treat influenza can be an important alternative for those who lack access to influenza vaccines or antiviral drugs. Lactobacillus plantarum (Lp) is one of many lactic acid bacteria that grow in 'kimchi', an essential part of Korean meal, and several strains of Lp reportedly show protective effects against influenza. Using heat-killed Lp (nF1) isolated from kimchi, which is known for its immunomodulatory effects, we investigated whether regular oral intake of nF1 could influence the outcome of influenza virus infection in a mouse model. In a lethal challenge with influenza A (H1N1 and H3N2 subtypes) and influenza B (Yamagata lineage) viruses, daily oral administration of nF1 delayed the mean number of days to death of the infected mice and resulted in increased survival rates compared with those of the non-treated mice. Consistent with these observations, nF1 treatment also significantly reduced viral replication in the lungs of the infected mice. Taken together, our results might suggest the remedial potential of heatkilled Lactobacillus probiotics against influenza.

Effects of Lactobacillus plantarum and Leuconostoc mesenteroides Probiotics on Human Seasonal and Avian Influenza Viruses.

Influenza viruses that cause recurrent seasonal epidemics to humans can be controlled with vaccine and antiviral therapy. However, the medical treatments often exhibit limited efficacy in the elderly or immunosuppressed individuals. In these cases, daily uptake of probiotic microbes may be an option to bring in health benefits against influenza. Here, we demonstrate the effects of probiotics Lactobacillus plantarum (Lp) and Leuconostoc mesenteroides (Lm) against seasonal and avian influenza viruses. As assessed by the plaque size reduction of human H1N1 and avian influenza H7N9 viruses, including green fluorescent protein-tagged H1N1 strain in cells, the selected Lp and Lm strains restrained viral replication in mouse lungs with statistical significance. Against lethal viral challenge, the Lp and Lm strains exhibited their beneficial effects by increasing the mean days and rates of survival of infected mice. These results suggest that, despite rather narrow ranges of protective efficacy, the dietary supplement of Lactobacillus and Leuconostoc probiotics may promote health benefits against influenza.

Single PA mutation as a high yield determinant of avian influenza vaccines.

Human infection with an avian influenza virus persists. To prepare for a potential outbreak of avian influenza, we constructed a candidate vaccine virus (CVV) containing hemagglutinin (HA) and neuraminidase (NA) genes of a H5N1 virus and evaluated its antigenic stability after serial passaging in embryonated chicken eggs. The passaged CVV harbored the four amino acid mutations (R136K in PB2; E31K in PA; A172T in HA; and R80Q in M2) without changing its antigenicity, compared with the parental CVV. Notably, the passaged CVV exhibited much greater replication property both in eggs and in Madin-Darby canine kidney and Vero cells. Of the four mutations, the PA E31K showed the greatest effect on the replication property of reverse genetically-rescued viruses. In a further luciferase reporter, mini-replicon assay, the PA mutation appeared to affect the replication property by increasing viral polymerase activity. When applied to different avian influenza CVVs (H7N9 and H9N2 subtypes), the PA E31K mutation resulted in the increases of viral replication in the Vero cell again. Taken all together, our results suggest the PA E31K mutation as a single, substantial growth determinant of avian influenza CVVs and for the establishment of a high-yield avian influenza vaccine backbone.

Adaptive mutations of neuraminidase stalk truncation and deglycosylation confer enhanced pathogenicity of influenza A viruses.

It has been noticed that neuraminidase (NA) stalk truncation has arisen from evolutionary adaptation of avian influenza A viruses (IAVs) from wild aquatic birds to domestic poultry. We identified this molecular alteration after the adaptation of a 2009 pandemic H1N1 virus (pH1N1) in BALB/c mice. The mouse-adapted pH1N1 lost its eight consecutive amino acids including one potential N-linked glycosite from the NA stalk region. To explore the relationship of NA stalk truncation or deglycosylation with viral pathogenicity changes, we generated NA stalk mutant viruses on the pH1N1 backbone by reverse genetics. Intriguingly, either NA stalk truncation or deglycosylation changed pH1N1 into a lethal virus to mice by resulting in extensive pathologic transformation in the mouse lungs and systemic infection affecting beyond the respiratory organs in mice. The increased pathogenicity of these NA stalk mutants was also reproduced in ferrets. In further investigation using a human-infecting H7N9 avian IAV strain, NA stalk truncation or deglycosylation enhanced the replication property and pathogenicity of H7N9 NA stalk mutant viruses in the same mouse model. Taken together, our results suggest that NA stalk truncation or deglycosylation can be the pathogenic determinants of seasonal influenza viruses associated with the evolutionary adaptation of IAVs.

Phylogenetic relationships of the HA and NA genes between vaccine and seasonal influenza A(H3N2) strains in Korea.

Seasonal influenza is caused by two influenza A subtype (H1N1 and H3N2) and two influenza B lineage (Victoria and Yamagata) viruses. Of these antigenically distinct viruses, the H3N2 virus was consistently detected in substantial proportions in Korea during the 2010/11-2013/14 seasons when compared to the other viruses and appeared responsible for the influenza-like illness rate peak during the first half of the 2011/12 season. To further scrutinize possible causes for this, we investigated the evolutionary and serological relationships between the vaccine and Korean H3N2 strains during the 2011/12 season for the main antigenic determinants of influenza viruses, the hemagglutinin (HA) and neuraminidase (NA) genes. In the 2011/12 season, when the number of H3N2 cases peaked, the majority of the Korean strains did not belong to the HA clade of A/Perth/16/2009 vaccine, and no Korean strains were of this lineage in the NA segment. In a serological assay, post-vaccinated human sera exhibited much reduced hemagglutination inhibition antibody titers against the non-vaccine clade Korean H3N2 strains. Moreover, Korean strains harbored several amino acid differences in the HA antigenic sites and in the NA with respect to vaccine lineages during this season. Of these, the HA antigenic site C residues 45 and 261 and the NA residue 81 appeared to be the signatures of positive selection. In subsequent seasons, when H3N2 cases were lower, the HA and NA genes of vaccine and Korean strains were more phylogenetically related to each other. Combined, our results provide indirect support for using phylogenetic clustering patterns of the HA and possibly also the NA genes in the selection of vaccine viruses and the assessment of vaccine effectiveness.

Reassortment compatibility between PB1, PB2, and HA genes of the two influenza B virus lineages in mammalian cells.

In addition to influenza A subtypes, two distinct lineages of influenza B virus also cause seasonal epidemics to humans. Recently, Dudas et al. have done evolutionary analyses of reassortment patterns of the virus and suggested genetic lineage relationship between PB1, PB2, and HA genes. Using genetic plasmids and reassortant viruses, we here demonstrate that a homologous lineage PB1-PB2 pair exhibits better compatibility than a heterologous one and that the lineage relationship between PB1 and HA is more important for viral replication than that between PB2 and HA. However, co-adaptation of PB1-PB2-HA genes appears to be affected by complete gene constellation.