Viral Diseases Research
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Development of a Universal Influenza Vaccine
Influenza is an infectious disease that greatly affects public health and socio-economics worldwide. Vaccines and antiviral drugs, as the primary methods of influenza management, are effective for infection prevention and illness treatment, respectively. Their availability plays a key role especially in the event of a pandemic. Current influenza vaccines are only effective against a narrow range of influenza subtypes or strains. Current trivalent (or quadrivalent) inactivated vaccines dominantly generate neutralizing antibodies (which determine the vaccine effectiveness) against the most variable surface antigen (HA1, the globular domain of hemagglutinin [HA]) of epidemic strains, resulting in annual vaccine strain selection. Many studies are targeting genetically and immunologically conserved proteins of the influenza virus such as the M2e and HA2 domains to develop broadly effective influenza vaccines that confer effective protection against various subtypes and strains (Figure.1).
Figure 1. Development of influenza universal vaccine candidates. Universal influenza vaccines based on HA stalk (A) and M2e (B).
Since 2016, the Division of Viral Disease Research has performed universal vaccine development and antiviral drug-related projects, titled “Development of pan-influenza vaccines and evaluation methods of immune response and alternative therapeutics against novel influenza viruses” to advance preparedness for pandemic and avian flu outbreaks in humans. The main project topic was the design and production of various influenza viral hemagglutinin (HA) stalk-based immunogens for the research and development of efficient pan-influenza vaccine candidates. The second topic, “Establishment of a neutralizing assay with influenza pseudo-typed viruses,” evaluated pan-flu vaccine efficacy by cloning pan-influenza immunogens, HA, and neuraminidase (NA) genes from swine H3N2 and avian H5N8 influenza viruses and expressing them in eukaryotic cells. In 2017, we designed a novel DNA vaccine from HA2 of an H1N1pdm09 strain by cloning the HA2 domain into a pCAG-EN vector. Furthermore, we observed that immunization with DNA vaccines induces protective immunity in mice against both homologous [A/Korea/01/2009 (H1N1)] and heterologous [A/Viet Nam/1194/2004 (H5N1) and A/Anhui/1/2013 (H7N9)] viruses (Figure 2). In addition, we evaluated the efficacy of pan-influenza vaccines by establishing a virus-like particle (VLP)-based neutralization assay as an alternative to current neutralizing assays for pandemic and avian flu.
Figure 2. Cross-protective immunity elicited by vaccination of H1 HA-DNA with AP against different influenza virus subtypes.
After the last DNA vaccine immunization, mice were intranasally infected with live (A) A/Korea/01-2-9/2009 (50mLD50), (B) A/VietNam/1194/2004 (50mLD50), or (C) A/Anhui/1/2012 (5mLD50). With alum phosphate, DNA vaccine carrying HA of influenza viruses conferred protective immunity to mice infected with the cognate and heterosubtypic influenza viruses.
Alternative influenza therapeutics research
To develop an alternative anti-influenza treatment, FDA-approved drugs and natural compound libraries were screened with divergent influenza A viruses. We identified eight influenza entry and six replication inhibitors. Among them, mycophenolic mofetil (MMF) demonstrated anti-viral activity with dual functions: inhibition of viral mRNA transcription and suppression of pro-inflammatory cytokine and chemokine expression against human-isolated H5N1 avian influenza virus in cells and mice (Figure 3).
Figure 3. MMF anti-viral activity and mechanism. (A) Half-maximal inhibitory concentrations of MMF or zanamivir in MDCK cells. B) Mice survival rate (%) of MMF and oseltamivir (OSE) treatment with three mouse LD50 of H5N1 virus infection. C) Anti-viral mechanism of MMF. MMF depleted guanosine: supplement of exogenous guanosine restored H5N1 viral NP, M1, and NS1 proteins.
In the near future, information and research products from these projects will be publicly shared through the publication, pending patents, and deposition of the research products for influenza pandemic preparedness. In addition, we will continuously develop efficient methods to evaluate pan-influenza vaccines and explore T-cell epitopes in terms of structural analysis to address low immunogenicity issues in current H7N9 vaccine development.
Development of a novel screening method to identify potent inhibitors of HIV Tat-mediated transcription
The TZM-bl cell line (also called the JC53BL-13 cell line) integrated with LTR-driven firefly luciferase (F-Luc) and lacZ genes allows quantitative analysis of HIV transcription activity based on Tat expression and/or HIV virion infection. Thus, this cell line has been used in many experiments to study HIV. To facilitate screening for inhibitors of HIV-1 Tat-mediated transcription, we developed a simplified single-cell system based on TZM-bl cells. Stable Tat expression can increase cytotoxicity, while transient Tat expression makes it difficult to screen putative candidates among a wide range of compounds; thus, a doxycycline (Dox)-inducible lentiviral system that constitutively expressed a tetracycline-controlled transactivator (tTA) tightly regulated by Dox allowed conditional expression of Tat. TZM-bl/Tat cells were designed by inserting lentiviral cassettes for Dox-inducible Tat expression, in which Tat expression was accompanied by the expression of F-Luc in the presence of Dox. Long terminal repeat (LTR)-reporter assay systems combined with Tat expression have been used to identify numerous inhibitors of Tat-induced transcription but these systems often lead to nonspecific inhibitors that affect general transcription without cytotoxicity. Thus, to minimize the off-target effects of reporter systems, TZM-bl/Tat/Rluc cells were designed by inserting a lentiviral cassette for Dox-inducible Renilla luciferase (R-Luc) expression into TZM-bl/Tat, which simultaneously expressed R-Luc and Tat in the presence of Dox. R-Luc activity indicated the general transcriptional activity and the relative Tat expression after Dox treatment. In this new scheme, when the candidate Tat-mediated transcription inhibitor was present in the medium, Tat-induced F-Luc activity could be turned off, and the R-Luc activity indicated whether the candidate had an off-target effect (Figure 4).
Figure 4. Scheme of the simplified system for screening Tat-mediated transcription inhibitors. Stable TZM-bl cells integrated with LTR-driven firefly luciferase (F-Luc) were serially selected with two doxycycline (Dox)-inducible lentiviral cassettes. The cassettes were constructed with Tat and the Renilla luciferase (R-Luc) gene downstream promoter regulated by the tetracycline-controlled transactivator (tTA), which was activated by treatment with Dox. TZM-bl/Tat/R-Luc cells treated with Dox expressed both Tat and R-Luc from the two lentiviral cassettes. The expressed Tat subsequently promoted the expression of F-Luc from the HIV-LTR promoter. The expression level of R-Luc indicated the cellular transcriptional activity to estimate the Tat expression level after treatment with Dox. If the cells were treated with a Tat-specific inhibitor in the presence of Dox, the Tat-induced F-Luc activity could be switched off without altering the R-Luc activity.
Epigenetic regulation of HIV-1 latency
HIV-1 reservoirs are a major obstacle to HIV-1 elimination in patients because the virus can reactivate when antiretroviral therapy (ART) is stopped. Histone modifications such as acetylation and methylation play a critical role in the organization of chromatin domains and the up- or down-regulation of gene expression. Although many studies have reported the strong involvement of an epigenetic mechanism in the maintenance of HIV-1 transcriptional latency, neither the epigenetic control of viral replication nor how HIV-1 latency is maintained is fully understood. We re-analysed high-throughput parallel DNA sequencing (ChIP-seq) data from previous work to investigate the effect of histone modifications H3K4me3 and H3K9ac on HIV-1 latency in terms of chromosome distribution. The outputs of ChIP-seq from uninfected CD4+ T cell lines and HIV-1 latently-infected cells were aligned to hg18 using bowtie and analysed using various software packages. Chromosomes 16, 17, 19, and 22 were significantly enriched for histone modifications in both decreased and increased islands (Figure 5A-C). In the same chromosomes in HIV-1 latently-infected cells, 38 decreased and 41 increased islands from common islands of H3K4me3 and H3K9ac were selected for functional annotation. In Gene Ontology analysis, the 38 genes associated with decreased islands were involved in the regulation of biological processes, regulation of cellular processes, biological regulation, and purinergic receptor signalling pathways, while the 41 genes associated with increased islands were involved in nucleic acid binding, calcium-activated cation channel activity, DNA binding, and zinc ion binding. In Pathway Commons analysis, the 38 genes were strongly involved in the p63 transcription factor network, while the 41 genes were involved in the RNA polymerase III transcription termination pathway. Several genes such as Nuclear factor IX (NFIX) and TNF receptor association factor 4 (TRAF4) were selected as candidate genes for HIV latency (Figure 5D and E). NFIX was highly expressed in HIV-1 latently-infected cell lines and showed a dramatic reduction in expression after phorbol-13-myristate-12-acetate (PMA) treatment. These results show that the unique enrichment of histone modifications and the linked genes in specific chromosomes might play a critical role in the establishment and maintenance of HIV-1 latency.
Figure 5. Identification of novel genes associated with HIV-1 latency by analysis of histone modifications. (A and B) Venn diagram of differentially enriched H3K4me3 and H3K9ac islands in chromosomes 16, 17, 19, and 22 of HIV-1 latently-infected cells. (C) Human chromosome map showing the localization of 38 decreased and 41 increased gene islands. (D) A visualization of the interactions of TOP2A, ITGB2, TRAF4, and SEC14L2 with NFIC and NFIX by Pathway Commons Network Visualizer (PCViz). The neighbourhood in query types shows the overall frequency of alteration for each gene in the network and the paths between query types shows the direct interactions of each gene in the network. (E) The result of real-time polymerase chain reactions (PCR) with and without PMA treatment in HIV-1 latently-infected cells.
ACK1 inhibits hepatitis B virus replication through down-regulation of viral enhancer activity
Persistent hepatitis B virus (HBV) infection is a major risk factor for liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Antiviral treatment for chronic hepatitis B (CHB) improves the outcome of liver disease and prevents HCC development. Currently, several nucleot(s)ide analogues including lamivudine (LMV), adefovir (ADV), entecavir (ETV), and tenofovir (TDF) have been approved for the treatment of CHB. However, long-term treatment with nucleot(s)ide analogues has been associated with drug resistance and viral breakthrough. Thus, the development of novel drugs targeting various steps of the HBV life cycle is necessary. Activated cdc42-associated kinase 1 (ACK1), also known as tyrosine kinase non-receptor 2 (TNK2), is a ubiquitously expressed non-receptor tyrosine kinase in various tissues. Several studies reported that ACK1 regulates molecules implicated in HBV replication, including Src, AKT, androgen receptors, and MAPK. However, the antiviral effect of ACK1 on HBV remains poorly understood. In this study, the effect of ACK1 on the HBV life cycle was investigated by ectopic expression in HepG2 and HepG2-ACK1 stable cell lines. Secretory HBeAg and HBsAg levels were measured in culture media and HBV DNA level was assessed by Southern blot. In addition, HBV enhancer activities (Enh I, Enh II/Cp) were determined using a reporter assay. We found that ACK1 suppressed the secretion of HBeAg/ HBsAg and HBV replication in a dose-dependent manner. ACK1 inhibited HBV RNA through down-regulation of HBV Enh I and Enh II/Cp activities. The molecular mechanism of the inhibitory effect of ACK1 against HBV is the suppression of hepatocytes nuclear factor 4 alpha (HNF4α) expression binding to HBV enhancers, eventually leading to decreased viral enhancer activity. Finally, we demonstrated that ACK1 inhibited HBV replication at the transcriptional level (Figure 6). Our findings suggest a novel ACK1 antiviral function as a potential modulator of host immune response for anti-HBV activity.
Figure 6. ACK1 inhibits HBV replication at the transcriptional level. (A) Effect of ACK1 on HBV replication. The indicated plasmids were transfected into HepG2 cells grown in 6-well plates and the cells were harvested 3 days later. M, Mock; +, 1 µg; ++, 2 µg; ds DNA, double-stranded HBV DNA; and ss DNA, single-stranded HBV DNA. (B) The secretion of HBeAg/HBsAg in HepG2 cell supernatant. The values represent the mean ± SD calculated from three independent experiments. (C) Establishment of ACK1 stable HepG2 cells. pIRES-ACK1 plasmids were transfected into HepG2 cells and ACK1-stable cells were selected with puromycin (1 µg/mL) for two weeks. (D) Effect of ACK1 on HBV transcription. Real-time PCR was performed using an ABI 7500 instrument. (E) Relative luciferase activity of HBV enhancer I and II in ACK1-stable HepG2 cells. (F and G) Effect of ACK1 on HNF4α mRNA in reverse transcription (RT)-PCR (F) and real-time PCR (G). Real-time PCR was performed using an ABI 7500 instrument. (H) Effect of ACK1 on HNF4α protein level.
Genetic variation and molecular epidemiology of hepatitis C viruses
Hepatitis C virus (HCV) infection, a major cause of acute hepatitis and chronic liver disease, is a widespread problem. HCV infections are treated using peginterferon plus ribavirin. The difficulties with this treatment included severe adverse events, inconvenience of injection, and the long treatment duration. The treatment paradigm is rapidly shifting from interferon-based therapy to interferon-free, direct-acting antivirals (DAAs) therapy, which leads to a sustained virological response (SVR) rate of 90% with minimal adverse events and a shorter treatment duration (12–24 weeks). The rapid replication rate of HCV and the low fidelity of its polymerase result in sequence variation in the HCV population, leading to quasi-species and the potential selection of drug-resistant mutations. The efficacy of DAAs is limited by the presence of mutations resulting in amino acid substitutions within the targeted proteins, which affect viral sensitivity to these compounds. HCV genotype is a critical factor in determining treatment regimen and durations. Resistance-associated variants (RAVs) can limit the efficacy of DAAs therapy and have been associated with an increased risk of therapy failure in some treatments. The sequence variability in HCV necessitates a subgenotype-specific PCR for successful amplification of RAV-containing regions. We developed a method for HCV cDNA synthesis and amplification of the NS5A and NS5B regions that are relevant for treatment decisions in a fast, reliable, and routine diagnostic setup. A flowchart summarizing the HCV NS5A and NS5B RT-PCR and the new primer sets is shown in Figure 7. The amplification efficiency of the NS5A and NS5B fragment differed according to the primer used for RT-PCR (Figure 8). In this study, we developed an RT-PCR method for HCV NS5A and NS5B synthesis. Using this method, amplicons covering NS5A and NS5B were successfully amplified from HCV-infected clinical samples to assess resistance.
Figure 7. Flowchart of NS5A and NS5B RT-PCR processes and primers.
Figure 8. Synthesis efficiency of NS5A (A) and NS5B (B) fragments using a new primer set. The sensitivity of PCR was enhanced by the new primer set. Fragment sizes: NS5A = 1489 bp, NS5B = 1790 bp.