Effect of trabeculectomy on the accuracy of intraocular lens calculations in patients with open-angle glaucoma.

BACKGROUND: The objective of the study is to examine the effect of trabeculectomy on intraocular lens power calculations in patients with open-angle glaucoma (OAG) undergoing cataract surgery. DESIGN: The design is retrospective data analysis. PARTICIPANTS: There are a total of 55 eyes of 55 patients with OAG who had a cataract surgery alone or in combination with trabeculectomy. METHODS: We classified OAG subjects into the following groups based on surgical history: only cataract surgery (OC group), cataract surgery after prior trabeculectomy (CAT group), and cataract surgery performed in combination with trabeculectomy (CCT group). MAIN OUTCOME MEASURES: Differences between actual and predicted postoperative refractive error. RESULTS: Mean error (ME, difference between postoperative and predicted SE) in the CCT group was significantly lower (towards myopia) than that of the OC group (P = 0.008). Additionally, mean absolute error (MAE, absolute value of ME) in the CAT group was significantly greater than in the OC group (P = 0.006). Using linear mixed models, the ME calculated with the SRK II formula was more accurate than the ME predicted by the SRK T formula in the CAT (P = 0.032) and CCT (P = 0.035) groups. CONCLUSIONS: The intraocular lens power prediction accuracy was lower in the CAT and CCT groups than in the OC group. The prediction error was greater in the CAT group than in the OC group, and the direction of the prediction error tended to be towards myopia in the CCT group. The SRK II formula may be more accurate in predicting residual refractive error in the CAT and CCT groups.

Cloning, expression, and characterization of thermophilic L-asparaginase from Thermococcus kodakarensis KOD1.

The present study demonstrates cloning, expression, and characterization of hyperthermostable L-asparaginase from Thermococcus kodakarensis KOD1 in Escherichia coli BLR(DE3). The recombinant 6× His-tagged protein L-asparaginase from T. kodakarensis (TkAsn), was purified to homogeneity by heat treatment followed by affinity chromatography using a nickel-nitrilotriacetic acid (Ni-NTA) column. The molecular mass of the purified enzyme was found to be approximately 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzymatic properties, such as optimum temperature and pH, were 90 °C and 8.0, respectively. Its appearent Km , Vmax , and Kcat values were 2.6 mM, 1121 µmol min(-1) mg(-1) , and 694 S(-1) , respectively. The enzyme displayed high thermal stability at optimum temperature with an insignificant loss in enzymatic activity, retaining almost 90% of its activity over a time period of 32 h. The relative activity of the enzyme was significantly inhibited by the supplementation of Cu(2+) and Ni(2+) ions, while moderately inhibited by other ions. In contrast, Mg(2+) ions enhanced the relative activity compared to the control. The acrylamide contents in baked dough were reduced to sixty percent after treatment with recombinant TkAsn as compared to the untreated control. Results of the present study revealed that the enzyme was highly active at broader range of temperatures and pH, which reflect the potential of recombinant TkAsn in the food processing industry. In addition, the high thermal stability of the enzyme may facilitates its handling, storage, and transportation.

The Effect of Organic Vegetable Mixed Juice on Blood Circulation and Intestine Flora: Randomized, Double-Blinded, Placebo-Controlled Clinical Trial.

Epidemiological evidence suggests that fruit and vegetable intake significantly positively affects cardiovascular health. Since vegetable juice is more accessible than raw vegetables, it attracts attention as a health functional food for circulatory diseases. Therefore, this study measured blood lipids, antioxidants, blood circulation indicators, and changes in the microbiome to confirm the effect of organic vegetable mixed juice (OVJ) on improving blood circulation. This 4-week, randomized, double-blinded, placebo-controlled study involved adult men and women with borderline total cholesterol (TC) and low-density lipoprotein (LDL) levels. As a result, blood lipid profile indicators, such as TC, triglycerides, LDL cholesterol, and apolipoprotein B, decreased (p < 0.05) in the OVJ group compared with those in the placebo group. Additionally, the antioxidant biomarker superoxide dismutase increased (p < 0.05). In contrast, systolic and diastolic blood viscosities, as blood circulation-related biomarkers, decreased (p < 0.05) in the OVJ group compared with those in the placebo group. After the intervention, a fecal microbiome analysis confirmed differences due to changes in the intestinal microbiome composition between the OVJ and placebo groups. In conclusion, our research results confirmed that consuming OVJ improves blood circulation by affecting the blood lipid profile, antioxidant enzymes, and microbiome changes.

Cold-adapted pandemic 2009 H1N1 influenza virus live vaccine elicits cross-reactive immune responses against seasonal and H5 influenza A viruses.

The rapid transmission of the pandemic 2009 H1N1 influenza virus (pH1N1) among humans has raised the concern of a potential emergence of reassortment between pH1N1 and highly pathogenic influenza strains, especially the avian H5N1 influenza virus. Here, we report that the cold-adapted pH1N1 live attenuated vaccine (CApH1N1) elicits cross-reactive immunity to seasonal and H5 influenza A viruses in the mouse model. Immunization with CApH1N1 induced both systemic and mucosal antibodies with broad reactivity to seasonal and H5 strains, including HAPI H5N1 and the avian H5N2 virus, providing complete protection against heterologous and heterosubtypic lethal challenges. Our results not only accentuate the merit of using live attenuated influenza virus vaccines in view of cross-reactivity but also represent the potential of CApH1N1 live vaccine for mitigating the clinical severity of infections that arise from reassortments between pH1N1 and highly pathogenic H5 subtype viruses.

Effects of Plant-Based Extract Mixture on Alcohol Metabolism and Hangover Improvement in Humans: A Randomized, Double-Blind, Paralleled, Placebo-Controlled Clinical Trial.

Hangovers are uncomfortable physiological symptoms after alcohol consumption caused by acetaldehyde, a toxic substance in which alcohol is metabolized by alcohol dehydrogenase (ADH). Rapid alcohol and acetaldehyde decomposition are essential to alleviate alcohol handling symptoms. This study investigated the effects of HY_IPA combined with Mesembryanthemum crystallinum, Pueraria lobata flower, and Artemisia indica on alleviating hangovers. A randomized, double-blind, parallel-group, placebo-controlled clinical study was conducted on 80 individuals with hangover symptoms. Alcohol intake was 0.9 g/bw with 40% whiskey, adjusted proportionately to body weight. The Acute Hangover Scale total score was 5.24 ± 5.78 and 18.54 ± 18.50 in the HY_ IPA and placebo groups, respectively (p < 0.0001). All nine indicators of the hangover symptom questionnaire were significantly improved in the HY_IPA group (p < 0.01). Blood alcohol and acetaldehyde concentrations rapidly decreased from 30 min in the HY_IPA group (p < 0.05). ADH and acetaldehyde dehydrogenase (ALDH) activities in the blood of the HY_IPA group were significantly higher than those in the placebo group at 0, 1, and 2 h after alcohol consumption (p < 0.01). The rapid hangover relief was due to increased ADH and ALDH. Therefore, HY_IPA effectively relieves hangover symptoms by decomposing alcohol and acetaldehyde when consumed before alcohol consumption.

Protective Effects of High-Fat Diet against Murine Colitis in Association with Leptin Signaling and Gut Microbiome.

Inflammatory bowel disease (IBD) is characterized by chronic intestinal-tract inflammation with dysregulated immune responses, which are partly attributable to dysbiosis. Given that diet plays a critical role in IBD pathogenesis and progression, we elucidated the effects of a high-fat diet (HFD) feeding on IBD development in relation to immune dysfunction and the gut microbiota. Five-week-old male C57BL/6J mice were fed either a normal diet (ND) or HFD for 14 weeks. The animals were further divided into ND, ND+ dextran sulfate sodium (DSS), HFD, and HFD+DSS treatment groups. The HFD+DSS mice exhibited lower body weight loss, lower disease activity index, longer colon length, and increased tight-junction protein expression and goblet-cell proportions compared with the ND+DSS mice. The T helper (h)1 and Th17 cell populations and pro-inflammatory cytokines involved in colitis pathogenesis were significantly more reduced in the HFD+DSS mice than in the ND+DSS mice. The HFD+DSS mice showed significantly increased serum leptin concentrations, colonic leptin receptor expression, enhanced anti-apoptotic AKT expression, and reduced pro-apoptotic MAPK and Bax expression compared with the ND+DSS mice, suggesting the involvement of the leptin-mediated pathway in intestinal epithelial cell apoptosis. The alterations in the gut-microbiota composition in the HFD+DSS group were the opposite of those in the ND+DSS group and rather similar to those of the ND group, indicating that the protective effects of HFD feeding against DSS-induced colitis are associated with changes in gut-microbiota composition. Overall, HFD feeding ameliorates DSS-induced colitis and colonic mucosal damage by reinforcing colonic barrier function and regulating immune responses in association with changes in gut-microbiota composition.

Immunogenicity of candidate SARS-CoV-2 DNA vaccines based on the spike protein.

Coronavirus disease 2019 caused by the novel human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently a major threat to public health worldwide. To deal with the needs of vaccine, we developed four DNA vaccine candidates against SARS-CoV-2, based on the full-length spike (S) or truncated S protein. Following mice vaccination, we measured T-cell response and antigen-specific neutralizing antibody (NAb) titer. All four candidates induced humoral immune responses, including elevated levels of total IgG and NAbs, and cell-mediated immune responses, including multiple cytokine expression. However, the full-length S DNA vaccine enhanced the immune responses most significantly. We then evaluated its appropriate antigen dose and vaccination schedule. Although all immunized groups showed higher immune response than the control group, inoculation with 50 µg antigen led to the highest NAb titer. Immunity was significantly increased after the third inoculation. Thus, the full-length S DNA vaccine can potentially prevent SARS-CoV-2 infection.

Long-Term Follow-Up of Patients with Neck Pain Associated with Ossification of the Posterior Longitudinal Ligament Treated with Integrative Complementary and Alternative Medicine: A Retrospective Analysis and Questionnaire Survey.

Purpose: Integrative Korean medicine treatment (KMT) is a conservative treatment approach for the ossification of the posterior longitudinal ligament (OPLL) in Korea; nonetheless, relevant studies focusing on KMT for OPLL are lacking. A multicenter retrospective analysis of patient medical records and a questionnaire survey were conducted to investigate the effectiveness of integrative KMT in patients with OPLL treated for neck pain. Patients and Methods: A total of 78 inpatients radiologically diagnosed with OPLL and treated for neck pain at four Korean medicine hospitals from April 1, 2016, to December 31, 2019, were enrolled. The primary index was an improvement in the numeric rating scale (NRS) score for neck pain, whereas the secondary outcome indices were improvements in the NRS score for arm pain, neck disability index (NDI) score, and EuroQol 5-dimension 5-level (EQ-5D-5L) score. Results: At discharge, the NRS score for neck pain, NRS score for arm pain, and NDI score decreased by 2.47 (95% confidence interval [CI], -2.81 to -2.14), 1.32 (95% CI, -1.73 to -0.91), and 16.02 (95% CI, -18.89 to -13.15), respectively, as compared with the scores at admission (p < 0.001). The EQ-5D-5L score increased by 0.12 (95% CI, 0.09 to 0.16) as compared with the score at admission (p < 0.001). This trend was also evident during follow-up. With respect to Patient Global Impression of Change evaluation, 33 (61.1%) patients claimed to have very much improved, whereas 17 (31.5%) patients reported to have much improved. Conclusion: Inpatients with OPLL who received integrative KMT showed improvements in neck pain, arm pain, the NDI, and quality of life, which were retained throughout the follow-up period.

Optimization of Zika DNA vaccine by delivery systems.

In our previous study, we designed and evaluated the efficacy of six DNA vaccine candidates based on the E protein of Zika virus (ZIKV). To optimize the DNA vaccine, we inoculated C57BL/6 and IFNAR1- mice with the vaccine candidate expressing tandem repeated ZIKV envelope domain III (ED III × 3) doses; 50 µg by intramuscular (IM), jet injection (JET), or electroporation (EP) routes. Results showed that vaccination by all routes induced humoral and cellular immunity. Among them, EP induced robust ZIKV E specific-total IgG and neutralizing antibodies, as well as T cell responses. Additionally, EP showed superior protective efficacy against the ZIKV Brazil strain compared to the IM and JET routes. Finally, in the dose optimization test of EP route, cellular immunity of 50 µg was induced a significant level than other dose groups. These results showed that the EP delivery system enhanced the potential immunogenicity and protective efficacy of DNA vaccines.

Development of an attenuated smallpox vaccine candidate: The KVAC103 strain.

Smallpox, a disease caused by the variola virus, is one of the most dangerous diseases and had killed numerous people before it was eradicated in 1980. However, smallpox has emerged as the most threatening bio-terrorism agent; as the first- and second-generation smallpox vaccines have been controversial and have caused severe adverse reactions, new demands for safe smallpox vaccines have been raised and some attenuated smallpox vaccines have been developed. We have developed a cell culture-based highly attenuated third-generation smallpox vaccine candidate KVAC103 strain by 103 serial passages of the Lancy-Vaxina strain derived from the Lister in Vero cells. Several clones were selected, taking into consideration their shape, size, and growth rate in mammalian cells. The clones were then inoculated intracerebrally in suckling mice to test for neurovirulence by observing survival. Protective immune responses in adult mice were examined by measuring the levels of neutralization antibodies and IFN-γ expression. Among several clones, clone 7 was considered the best alternative candidate because there was no mortality in suckling mice against a lethal challenge. In addition, enhanced neutralizing antibodies and T-cell mediated IFN-γ production were observed in clone 7-immunized mice. Clone 7 was named "KVAC103" and was used for the skin toxicity test and full-genome analysis. KVAC103-inoculated rabbits showed reduced skin lesions compared to those inoculated with the Lister strain, Lancy-Vaxina. A whole genome analysis of KVAC103 revealed two major deleted regions that might contribute to the reduced virulence of KVAC103 compared to the Lister strain. Phylogenetic inference supported the close relationship with the Lister strain. Collectively, our data demonstrate that KVAC103 holds promise for use as a third-generation smallpox vaccine strain due to its enhanced safety and efficacy.

Enhanced effect of modified Zika virus E antigen on the immunogenicity of DNA vaccine.

It has been reported worldwide that the Zika virus (ZIKV) could be transmitted through placentas and sexual contact. ZIKV can also cause Guillain-Barre syndrome, microcephaly and neurological abnormalities. However, there are no approved vaccines available. We constructed six DNA vaccine candidates and tested the immunogenicity. Tandem repeated envelope domain Ⅲ (ED Ⅲ × 3) induced highly total IgG and neutralization antibody, as well as CD8+ T cell responses. Also, stem region-removed envelope (E ΔSTEM) elicited a robust production of IFN-γ in mice. To examine in vivo protection, we used mice treated with an IFNAR1 blocking antibody before and after the challenge. Vaccination with the two candidates led to a decline in the level of viral RNAs in organs. Moreover, the sera from the vaccinated mice did not enhance the infection of Dengue virus in K562 cells. These findings suggest the potential for the development of a novel ZIKV DNA vaccine.

Evaluation of green tea extract as a safe personal hygiene against viral infections.

BACKGROUND: Viral infections often pose tremendous public health concerns as well as economic burdens. Despite the availability of vaccines or antiviral drugs, personal hygiene is considered as effective means as the first-hand measure against viral infections. The green tea catechins, in particular, epigallocatechin-3-gallate (EGCG), are known to exert potent antiviral activity. In this study, we evaluated the green tea extract as a safe personal hygiene against viral infections. RESULTS: Using the influenza virus A/Puerto Rico/8/34 (H1N1) as a model, we examined the duration of the viral inactivating activity of green tea extract (GTE) under prolonged storage at various temperature conditions. Even after the storage for 56 days at different temperatures, 0.1% GTE completely inactivated 106 PFU of the virus (6 log10 reduction), and 0.01% and 0.05% GTE resulted in 2 log10 reduction of the viral titers. When supplemented with 2% citric acid, 0.1% sodium benzoate, and 0.2% ascorbic acid as anti-oxidant, the inactivating activity of GTE was temporarily compromised during earlier times of storage. However, the antiviral activity of the GTE was steadily recovered up to similar levels with those of the same concentrations of GTE without the supplements, effectively prolonging the duration of the virucidal function over extended period. Cryo-EM and DLS analyses showed a slight increase in the overall size of virus particles by GTE treatment. The results suggest that the virucidal activity of GTE is mediated by oxidative crosslinking of catechins to the viral proteins and the change of physical properties of viral membranes. CONCLUSIONS: The durability of antiviral effects of GTE was examined as solution type and powder types over extended periods at various temperature conditions using human influenza A/H1N1 virus. GTE with supplements demonstrated potent viral inactivating activity, resulting in greater than 4 log10 reduction of viral titers even after storage for up to two months at a wide range of temperatures. These data suggest that GTE-based antiviral agents could be formulated as a safe and environmentally friendly personal hygiene against viral infections.

Cell-cultured, live attenuated, X-31ca-based H5N1 pre-pandemic influenza vaccine.

The manufacture of influenza vaccines has traditionally depended upon a method using embryonated hen's eggs. However, concerns regarding the potential shortage of the influenza substrate in the event of a pandemic has led to the development of cell culture-derived vaccines, which offers shorter lead-in times and greater production flexibility. We examined optimal conditions for the production of reassortant X-31ca-based H5N1 cold-adapted live attenuated influenza vaccine (rH5N1ca) cultured in mammalian cell lines. During ten passages in MDCK cells, the rH5N1ca vaccine maintained cold-adapted and temperature-sensitive phenotypes, and no mutations occurred in the hemagglutinin and neuraminidase surface antigens, demonstrating genetic and phenotypic stability. Single immunization in mice with the rH5N1ca induced robust antibody responses and protected the mice against lethal challenge. Stable maintenance of attenuation phenotypes and immunogenicity of the rH5N1ca from cell-culture suggest that they can be produced as a stockpile for pandemic preparedness as an alternative to current egg-based production.

Risk factors for visual field progression of normal-tension glaucoma in patients with myopia.

OBJECTIVE: To identify risk factors for visual field progression of normal-tension glaucoma (NTG) in patients with myopia. DESIGN: Longitudinal, observational study. PARTICIPANTS: Fifty-one eyes of 51 NTG patients with myopia (less than -0.75D based on spherical equivalence) who had undergone visual field (VF) testing at least once per year for ≥6 years between November 2005 and December 2013. METHODS: Progression was defined using event-based guided progression analysis. Risk factors were analyzed using the Cox proportional hazards model and further tested for independence in a multivariate model. RESULTS: The mean observation period was 7.0 ± 1.3 years, and 16 of 51 subjects showed progression. In the univariate analysis, abnormal retinal nerve fibre layer (RNFL) colour codes (yellow or red sector) at the 11, 10, and 7 o'clock positions on optical coherence tomography showed significant associations with the VF progression (p = 0.03, 0.03, and 0.01, respectively). In the final multivariate models, the abnormal RNFL colour code of the 7 o'clock sector (inferotemporal sector) was the only significant risk factor for progression (hazard ratio = 4.07 and 4.37; 95% CI, 1.11-14.92 and 1.27-15.04; p = 0.03 and 0.02, respectively). CONCLUSIONS: Inferotemporal RNFL thinning could be a risk factor for progression in NTG patients with myopia.

Influence of epiretinal membrane on the measurement of peripapillary retinal nerve fibre layer thickness using spectral-domain coherence tomography.

BACKGROUND/AIMS: To investigate the effect of epiretinal membrane (ERM) on peripapillary retinal nerve fibre layer (RNFL) thickness measurements using spectral-domain optical coherence tomography (Spectralis; Heidelberg Engineering). METHODS: A total of 134 patients with idiopathic ERM and 63 healthy controls were included in this observational comparative study. We categorised ERMs into three severity grades, based on retinal appearance in macular scans. All eyes with ERM were classified into two groups; those involving the peripapillary scan area (ERM+pp, n=68 eyes) and not involving the peripapillary scan area (ERM-pp, n=66 eyes) using the macular disc scan. Peripapillary RNFL thickness was compared between related subgroups as (ERM+pp) or (ERM-pp) group. RESULTS: Temporal peripapillary RNFL thickness was significantly greater in the ERM+pp group (109.44±22.91 µm), followed by the ERM-pp (82.60±11.77 µm, p<0.001) and control (75.42±10.49 µm, p<0.001) groups. Temporal peripapillary RNFL thickness significantly increased with ERM grade in the ERM+pp and ERM-pp groups (both p<0.001). The peripapillary RNFL thickness was overestimated (exceeded 99th percentile) in the temporal sector in 49 eyes (72.1%) in the ERM+pp group and in 5 eyes (7.6%) in the ERM-pp group. CONCLUSIONS: Temporal and global peripapillary RNFL thickness is significantly higher in eyes with ERM, especially when the ERM extends into the peripapillary area. However, some eyes with an ERM that does not involve the peripapillary scan area still show peripapillary RNFL thickening. Measured peripapillary RNFL thickness was significantly and positively correlated with ERM severity.

Enhancement of the safety of live influenza vaccine by attenuating mutations from cold-adapted hemagglutinin.

In our previous study, X-31ca-based H5N1 LAIVs, in particular, became more virulent in mice than the X-31ca MDV, possibly by the introduction of the surface antigens of highly pathogenic H5N1 influenza virus, implying that additional attenuation is needed in this cases to increase the safety level of the vaccine. In this report we suggest an approach to further increase the safety of LAIV through additional cold-adapted mutations in the hemagglutinin. The cold-adaptation of X-31 virus resulted in four amino acid mutations in the HA. We generated a panel of 7:1 reassortant viruses each carrying the hemagglutinins with individual single amino acid mutations. We examined their phenotypes and found a major attenuating mutation, N81K. This attenuation marker conferred additional temperature-sensitive and attenuation phenotype to the LAIV. Our data indicate that the cold-adapted mutation in the HA confers additional attenuation to the LAIV strain, without compromising its productivity and immune response.

Estimated Prevalence of Glaucoma in South Korea Using the National Claims Database.

Purpose. To estimate the prevalence of glaucoma and costs associated with glaucoma care in South Korea between 2008 and 2013 using the Korean national claims database. Design. Retrospective cross-sectional study from a national claims database. Methods. Patients who were diagnosed with glaucoma between 2008 and 2013 were retrospectively identified in the national claims database using glaucoma diagnostic codes. For each year, the prevalence of glaucoma and direct medical costs associated with glaucoma care were estimated. Result. The prevalence of glaucoma in patients ≥40 years of age increased from 0.79% in 2008 to 1.05% in 2013. The number of patients with glaucoma increased by 54% between 2008 and 2013 (9% average annual increase). The prevalence of glaucoma increased with age and was higher in males than in females. The cost to care for glaucoma patients increased from $16.5 million in 2008 to $29.2 million in 2013, which translated into an 81% increase over the 6 years examined (12.7% average annual increase). Conclusion. The estimated prevalence and socioeconomic burden of glaucoma have steadily increased each year in South Korea. Nevertheless, many glaucoma patients remain undiagnosed in the present study using national claims database.

Frequency, Type and Cause of Artifacts in Swept-Source and Cirrus HD Optical Coherence Tomography in Cases of Glaucoma and Suspected Glaucoma.

PURPOSE: To evaluate and compare the frequency, type and cause of imaging artifacts incurred when using swept-source optical coherence tomography (SS OCT) and Cirrus HD OCT in the same patients on the same day. MATERIALS AND METHODS: From left eye OCT results of 72 patients, disc area and macular area data could be compared between the two types of OCT. For each scan, the final printout report and source data were examined. For comparison between the two types of OCT, only source image data were used because of differences in the final printout report format. RESULTS: There were no significant differences in the artifact frequencies between the two groups in either area (disc area: 35.9% of SS OCT, 42.2% of Cirrus OCT, p = 0.523; Macular area: 24.2% of SS OCT, 22.7% of Cirrus OCT, p = 1.00). The overall results of artifact comparison between the two types of OCTs also showed no significant differences. Boundary misidentification was the most common type of artifact observed, and ocular pathology was the most common cause of artifact in both types of OCTs. Among ocular pathologies, the epiretinal membrane (ERM) was the most common cause of OCT artifact production in both types of OCTs. CONCLUSIONS: There was no significant difference in the frequency, type and cause of artifacts between SS OCT and Cirrus HD OCT. Artifacts in OCT can influence the interpretation of OCT results. In particular, ERM around the optic disc could contribute to OCT artifacts and should be considered in glaucoma diagnosis or during patient follow-up using OCT.

High-yield soluble expression of recombinant influenza virus antigens from Escherichia coli and their potential uses in diagnosis.

Although antiviral drugs and vaccines have been successful for mitigating influenza virus infections, the lack of general technical platform for the timely supply of soluble and highly purified influenza viral antigens presents a serious bottleneck for the subsequent analysis for the effective control of the viral disease. Using the Escherichia coli (E. coli) lysyl tRNA synthetase (LysRS) as a novel fusion partner, this study reports the soluble expression of influenza viral proteins in E. coli host, construction of antibody library against the virus, and detection of anti-influenza antibodies using the expressed viral antigens. When influenza A and B viral proteins were fused with the LysRS, the fusion proteins were expressed predominantly as soluble forms and their production yields were high enough to be amenable to immunization protocols in rabbits for antibody generation. The produced antibodies showed high level binding specificity against the respective viral proteins, with cross-reactivity across heterologous viruses within the same type of influenza viruses. In addition, LysRS-HA fusion protein could bind specifically to anti-HA antibodies in the virus-infected mouse serum in widely accepted two detection methods, Western blot and ELISA. These results present a convenient tool for the production of antibodies specific to the virus as well as the rapid detection of influenza viral infections, ultimately contributing to the control of influenza viruses including highly pathogenic H5N1, pandemic H1N1, or the recent H7N9 influenza viruses.

Protective efficacy in mice of monovalent and trivalent live attenuated influenza vaccines in the background of cold-adapted A/X-31 and B/Lee/40 donor strains.

Influenza virus continues to take a heavy toll on human health and vaccination remains the mainstay of efforts to reduce the clinical impact imposed by viral infections. Proven successful for establishing live attenuated vaccine donor strains, cold-adapted live attenuated influenza vaccines (CAIVs) have become an attractive modality for controlling the virus infection. Previously, we developed the cold-adapted strains A/X-31 and B/Lee/40 as novel donor strains of CAIVs against influenza A and B viruses. In this study, we investigated the protective immune responses of both mono- and trivalent vaccine formulations in the mouse model. Two type A vaccines and one type B vaccine against A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2), and B/Shangdong/7/97 in the background of the A/X-31 ca or B/Lee/40 ca were generated by a reassortment procedure and evaluated for their immunogenicity and protective efficacy. Each monovalent vaccine elicited high levels of serum antibodies and conferred complete protection against homologous wild type virus infection. As compared to the monovalent vaccines, trivalent formulation induced higher levels of type A-specific serum antibodies and slightly lower levels of type B-specific antibodies, suggesting an immunological synergism within type A viruses and an interference in the replication of type B virus. Relatively lower type B-specific immunogenicity in trivalent vaccine formulation could be effectively implemented by increasing the vaccine dose of influenza B virus. These results of immunogenicity, protection efficacy, and immunological synergism between type A vaccines provide an experimental basis for optimal composition of trivalent vaccines for subsequent developments of multivalent CAIVs against seasonal and pandemic influenza viruses.