Insights into the immune responses of SARS-CoV-2 in relation to COVID-19 vaccines.

The three types of approved coronavirus disease 2019 (COVID-19) vaccines that have been emergency-use listed (EUL) by the World Health Organization are mRNA vaccines, adenovirus-vectored vaccines, and inactivated vaccines. Canonical vaccine developments usually take years or decades to be completed to commercialization; however, the EUL vaccines being used in the current situation comprise several COVID-19 vaccine candidates applied in studies and clinical settings across the world. The extraordinary circumstances of the COVID-19 pandemic have necessitated the emergency authorization of these EUL vaccines, which have been rapidly developed. Although the benefits of the EUL vaccines outweigh their adverse effects, there have been reports of rare but fatal cases directly associated with COVID-19 vaccinations. Thus, a reassessment of the immunological rationale underlying EUL vaccines in relation to COVID-19 caused by SARSCOV-2 virus infection is now required. In this review, we discuss the manifestations of COVID-19, immunologically projected effects of EUL vaccines, reported immune responses, informed issues related to COVID-19 vaccination, and the potential strategies for future vaccine use against antigenic variants.

The Immune Correlates of Orthohantavirus Vaccine.

Zoonotic transmission of orthohantaviruses from rodent reservoirs to humans has been the cause of severe fatalities. Human infections are reported worldwide, but vaccines have been approved only in China and Korea. Orthohantavirus vaccine development has been pursued with no sense of urgency due to the relative paucity of cases in countries outside China and Korea. However, the orthohantaviruses continuously evolve in hosts and thus the current vaccine may not work as well against some variants. Therefore, a more effective vaccine should be prepared against the orthohantaviruses. In this review, we discuss the issues caused by the orthohantavirus vaccine. Given the pros and cons of the orthohantavirus vaccine, we suggest strategies for the development of better vaccines in terms of pandemic preparedness.

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.

Animal models for the risk assessment of viral pandemic potential.

Pandemics affect human lives severely and globally. Experience predicts that there will be a pandemic for sure although the time is unknown. When a viral epidemic breaks out, assessing its pandemic risk is an important part of the process that characterizes genomic property, viral pathogenicity, transmission in animal model, and so forth. In this review, we intend to figure out how a pandemic may occur by looking into the past influenza pandemic events. We discuss interpretations of the experimental evidences resulted from animal model studies and extend implications of viral pandemic potentials and ingredients to emerging viral epidemics. Focusing on the pandemic potential of viral infectious diseases, we suggest what should be assessed to prevent global catastrophes from influenza virus, Middle East respiratory syndrome coronavirus, dengue and Zika viruses.

Novel Small Molecule Targeting the Hemagglutinin Stalk of Influenza Viruses.

Combating influenza is one of the perennial global public health issues to be managed. Antiviral drugs are useful for the treatment of influenza in the absence of an appropriate vaccine. However, the appearance of resistant strains necessitates a constant search for new drugs. In this study, we investigated novel anti-influenza drug candidates using in vitro and in vivo assays. We identified anti-influenza hit compounds using a high-throughput screening method with a green fluorescent protein-tagged recombinant influenza virus. Through subsequent analyses of their cytotoxicity and pharmacokinetic properties, one candidate (IY7640) was selected for further evaluation. In a replication kinetics analysis, IY7640 showed greater inhibitory effects during the early phase of viral infection than the viral neuraminidase inhibitor oseltamivir. In addition, we observed that hemagglutinin (HA)-mediated membrane fusion was inhibited by IY7640 treatment, indicating that the HA stalk region, which is highly conserved across various (sub)types of influenza viruses, may be the molecular target of IY7640. In an escape mutant analysis in cells, amino acid mutations were identified at the HA stalk region of the 2009 pandemic H1N1 (pH1N1) virus. Even though the in vivo efficacy of IY7640 did not reach complete protection in a lethal challenge study in mice, these results suggest that IY7640 has potential to be developed as a new type of anti-influenza drug.IMPORTANCE Anti-influenza drugs with broad-spectrum efficacy against antigenically diverse influenza viruses can be highly useful when no vaccines are available. To develop new anti-influenza drugs, we screened a number of small molecules and identified a strong candidate, IY7640. When added at the time of or after influenza virus infection, IY7640 was observed to successfully inhibit or reduce viral replication in cells. We subsequently discovered that IY7640 targets the stalk region of the influenza HA protein, which exhibits a relatively high degree of amino acid sequence conservation across various (sub)types of influenza viruses. Furthermore, IY7640 was observed to block HA-mediated membrane fusion of H1N1, H3N2, and influenza B viruses in cells. Although it appears less effective against strains other than H1N1 subtype viruses in a challenge study in mice, we suggest that the small molecule IY7640 has potential to be optimized as a new anti-influenza drug.

One-step multiplex real-time RT-PCR for detection and typing of dengue virus.

Previous studies reported that severity of dengue is associated with multiple factors, including secondary infection, age, viral load and infecting serotype and genotype. In addition, other studies have reported that a dengue virus-2 (DENV-2) infection is associated with a prognosis of more severe clinical manifestations than DENV-1 and DENV-4 infections. For these reasons, the ability to identify the DENV serotypes is critical for optimal patient diagnosis and epidemiological studies. In this study, we developed a TaqMan probe-based, one-step real-time reverse transcriptase-polymerase chain reaction (RT-PCR) system for detection and serotyping DENV. Our linear dynamic range (101 to 107 copies/reaction) showed the R2 values of DENV-1, 2, 3 and 4 as 0.998, 0.998, 0.994, and 0.998, respectively. The detection limits of DENV-1, 2, 3, and 4, were 10 copies/reaction, 100 copies/reaction, 10 copies/reaction, and 100 copies/reaction, respectively. Specificity test results indicated that this system is specific for DENV-1, 2, 3, and 4 and does not react with other viruses. Finally, we validated our results with five different real-time PCR instruments. Our results showed that the Ct values of the four serotype templates were similar in five real-time PCR instruments. Thus, this system provides an accurate method for detection and serotyping of DENV, which can be applied in diagnostics, surveillance, and epidemiology. Dengue can be found in many nations with varying socioeconomic and monetary resources. The results of our validation analyses using five different real-time PCR instruments suggest that this method can easily and confidently be used world-wide.

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.

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.

Towards the Application of Human Defensins as Antivirals.

Defensins are antimicrobial peptides that participate in the innate immunity of hosts. Humans constitutively and/or inducibly express α- and ß-defensins, which are known for their antiviral and antibacterial activities. This review describes the application of human defensins. We discuss the extant experimental results, limited though they are, to consider the potential applicability of human defensins as antiviral agents. Given their antiviral effects, we propose that basic research be conducted on human defensins that focuses on RNA viruses, such as human immunodeficiency virus (HIV), influenza A virus (IAV), respiratory syncytial virus (RSV), and dengue virus (DENV), which are considered serious human pathogens but have posed huge challenges for vaccine development for different reasons. Concerning the prophylactic and therapeutic applications of defensins, we then discuss the applicability of human defensins as antivirals that has been demonstrated in reports using animal models. Finally, we discuss the potential adjuvant-like activity of human defensins and propose an exploration of the 'defensin vaccine' concept to prime the body with a controlled supply of human defensins. In sum, we suggest a conceptual framework to achieve the practical application of human defensins to combat viral infections.

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.

Original Antigenic Sin Response to RNA Viruses and Antiviral Immunity.

The human immune system has evolved to fight against foreign pathogens. It plays a central role in the body's defense mechanism. However, the immune memory geared to fight off a previously recognized pathogen, tends to remember an original form of the pathogen when a variant form subsequently invades. This has been termed 'original antigenic sin'. This adverse immunological effect can alter vaccine effectiveness and sometimes cause enhanced pathogenicity or additional inflammatory responses, according to the type of pathogen and the circumstances of infection. Here we aim to give a simplified conceptual understanding of virus infection and original antigenic sin by comparing and contrasting the two examples of recurring infections such as influenza and dengue viruses in humans.

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.

Inhibition of Pseudomonas aeruginosa with a recombinant RNA-based viral vector expressing human ß-defensin 4.

BACKGROUND: Harassed with extensive epithelial burn wounds, patients can be affected by complications, such as infection, hypovolemic shock, hypothermia, and respiratory failure. Immediate first aid and followed supportive cares are critical for the prevention of severe complications. However, secondary bacterial infection is hard to be controlled in burn patients, and Pseudomonas aeruginosa (P. aeruginosa) is one of the top listed pathogens perturbing burn wounds beyond the antibiotics spectrum. RESULTS: To find the way for efficacious protection from the pseudomonas-mediated complications in burn patients, we assessed the in vitro and in vivo inhibitory values of human ß-defensin 4 (hBD4), which is known as a member of the cationic, antimicrobial peptides found in human cells of many kinds. The Newcastle disease virus (NDV) was used as a viral vector for the expression of hBD4 in burn wounds. Expressed from the recombinant NDV (rNDV-hBD4), hBD4 effectively inhibited the pseudomonal growths in cell culture media. In a mouse model, severely burn-injured skin was recovered by the direct installation of the rNDV-hBD4 infected cells in the burn wounds whereas that of control mice remained severely damaged. CONCLUSIONS: We suggest that the application of hBD4 may protect burn patients from secondary pseudomonal infection and provide a therapeutic potential for burn wound treatment.

The PDZ-binding motif of the avian NS1 protein affects transmission of the 2009 influenza A(H1N1) virus.

By nature of their segmented RNA genome, influenza A viruses (IAVs) have the potential to generate variants through a reassortment process. The influenza nonstructural (NS) gene is critical for a virus to counteract the antiviral responses of the host. Therefore, a newly acquired NS segment potentially determines the replication efficiency of the reassortant virus in a range of different hosts. In addition, the C-terminal PDZ-binding motif (PBM) has been suggested as a pathogenic determinant of IAVs. To gauge the pandemic potential from human and avian IAV reassortment, we assessed the replication properties of NS-reassorted viruses in cultured cells and in the lungs of mice and determined their transmissibility in guinea pigs. Compared with the recombinant A/Korea/01/2009 virus (rK09; 2009 pandemic H1N1 strain), the rK09/VN:NS virus, in which the NS gene was adopted from the A/Vietnam/1203/2004 virus (a human isolate of the highly pathogenic avian influenza H5N1 virus strains), exhibited attenuated virulence and reduced transmissibility. However, the rK09/VN:NS-PBM virus, harboring the PBM in the C-terminus of the NS1 protein, recovered the attenuated virulence of the rK09/VN:NS virus. In a guinea pig model, the rK09/VN:NS-PBM virus showed even greater transmission efficiency than the rK/09 virus. These results suggest that the PBM in the NS1 protein may determine viral persistence in the human and avian IAV interface.

The anti-influenza virus effect of Phellinus igniarius extract.

Herbal medicine has been used in the orient for thousands of years to treat large and small ailments, including microbial infections. Although there are treatments for influenza virus infection, there is no treatment for drug-resistant viruses. It is time that we explored and exploited the multi-component nature of herbal extracts as multi-drug combination therapies. Here, we present data on the anti-influenza virus effect of a medicinal mushroom, Phellinus igniarius. The P. igniarius water extract was effective against influenza A and B viruses, including 2009 pandemic H1N1, human H3N2, avian H9N2, and oseltamivir-resistant H1N1 viruses. Virological assays revealed that the extract may interfere with one or more early events in the influenza virus replication cycle, including viral attachment to the target cell. Therefore, our results provide new insights into the use of P. igniarius as an anti-influenza medicine.

Genetic requirement for hemagglutinin glycosylation and its implications for influenza A H1N1 virus evolution.

Influenza A virus has evolved and thrived in human populations. Since the 1918 influenza A pandemic, human H1N1 viruses had acquired additional N-linked glycosylation (NLG) sites within the globular head region of hemagglutinin (HA) until the NLG-free HA head pattern of the 1918 H1N1 virus was renewed with the swine-derived 2009 pandemic H1N1 virus. Moreover, the HA of the 2009 H1N1 virus appeared to be antigenically related to that of the 1918 H1N1 virus. Hence, it is possible that descendants of the 2009 H1N1 virus might recapitulate the acquisition of HA head glycosylation sites through their evolutionary drift as a means to evade preexisting immunity. We evaluate here the evolution signature of glycosylations found in the globular head region of H1 HA in order to determine their impact in the virulence and transmission of H1N1 viruses. We identified a polymorphism at HA residue 147 associated with the acquisition of glycosylation at residues 144 and 172. By in vitro and in vivo analyses using mutant viruses, we also found that the polymorphism at HA residue 147 compensated for the loss of replication, virulence, and transmissibility associated with the presence of the N-linked glycans. Our findings suggest that the polymorphism in H1 HA at position 147 modulates viral fitness by buffering the constraints caused by N-linked glycans and provide insights into the evolution dynamics of influenza viruses with implications in vaccine immunogenicity.

DBA/2 mouse as an animal model for anti-influenza drug efficacy evaluation.

Influenza viruses are seasonally recurring human pathogens. Vaccines and antiviral drugs are available for influenza. However, the viruses, which often change themselves via antigenic drift and shift, demand constant efforts to update vaccine antigens every year and develop new agents with broad-spectrum antiviral efficacy. An animal model is critical for such efforts. While most human influenza viruses are unable to kill BALB/c mice, some strains have been shown to kill DBA/2 mice without prior adaptation. Therefore, in this study, we explored the feasibility of employing DBA/2 mice as a model in the development of anti-influenza drugs. Unlike the BALB/c strain, DBA/2 mice were highly susceptible and could be killed with a relatively low titer (50% DBA/2 lethal dose = 10(2.83) plaque-forming units) of the A/Korea/01/2009 virus (2009 pandemic H1N1 virus). When treated with a neuraminidase inhibitor, oseltamivir phosphate, infected DBA/2 mice survived until 14 days post-infection. The reduced morbidity of the infected DBA/2 mice was also consistent with the oseltamivir treatment. Taking these data into consideration, we propose that the DBA/2 mouse is an excellent animal model to evaluate antiviral efficacy against influenza infection and can be further utilized for combination therapies or bioactivity models of existing and newly developed anti-influenza drugs.

The clamp loader assembles the beta clamp onto either a 3' or 5' primer terminus: the underlying basis favoring 3' loading.

Clamp loaders assemble sliding clamps onto 3' primed sites for DNA polymerases. Clamp loaders are thought to be specific for a 3' primed site, and unable to bind a 5' site. We demonstrate here that the Escherichia coli gamma complex clamp loader can load the beta clamp onto a 5' primed site, although with at least 20-fold reduced efficiency relative to loading at a 3' primed site. Preferential clamp loading at a 3' site does not appear to be due to DNA binding, as the clamp loader forms an avid complex with beta at a 5' site. Preferential loading at a 3' versus a 5' site occurs at the ATP hydrolysis step, needed to close the ring around DNA. We also address DNA structural features that are recognized for preferential loading at a 3' site. Although the single-stranded template strand extends in opposite directions from 3' and 5' primed sites, thus making it a favorite candidate for distinguishing between 3' and 5' sites, the single-strand polarity at a primed template junction does not determine 3' site selection for clamp loading. Instead, we find that clamp loader recognition of a 3' site lies in the duplex portion of the primed site, not the single-strand portion. We present evidence that the beta clamp facilitates its own loading specificity for a 3' primed site. Implications to eukaryotic clamp loader complexes are proposed.

Immunomodulatory roles of the carcinoembryonic antigen family of glycoproteins.

One of the most remarkable aspects of the immune system is its ability to fashion an immune response most appropriate to the activating stimulus. Although the immune system possesses a number of adaptations to accomplish this, an important theme is local immune regulation by site-specific expression of receptors and ligands. One family of molecules that is gaining attention as modulators of the immune system is the carcinoembryonic antigen cell-adhesion molecule family (CEACAM). Functionally, the carcinoembryonic antigen family can mediate cell-cell contact, host-pathogen interactions, and immune regulation. For example, biliary glycoprotein (CEACAM1) can have direct activity on T cells, leading to the inhibition of helper or cytotoxic T cell function. The expression of carcinoembryonic antigen (CEACAM5) on intestinal epithelial cells is involved in the activation of populations of regulatory CD8(+) T cells, while a distinct subset of regulatory CD8+ T cells is activated by nonspecific cross-reacting antigen (CEACAM6) on placental trophoblasts. Interestingly, the function and phenotype of these cells depend upon the specific member of the carcinoembryonic antigen family expressed, as well as the antigen-presenting molecule with which it associates. Thus, these glycoproteins comprise a family of molecules whose functions can depend on their nature and context.