Haryoung Poo

Mucosal Immunization with Surface-Displayed Severe Acute Respiratory Syndrome Coronavirus Spike Protein on Lactobacillus casei Induces Neutralizing Antibodies in Mice

ABSTRACT Induction of mucosal immunity may be important for preventing SARS-CoV infections. For safe and effective delivery of viral antigens to the mucosal immune system, we have developed a novel surface antigen display system for lactic acid bacteria using the poly-γ-glutamic acid synthetase A protein (PgsA) of Bacillus subtilis as an anchoring matrix. Recombinant fusion proteins comprised of PgsA and the Spike (S) protein segments SA (residues 2 to 114) and SB (residues 264 to 596) were stably expressed in Lactobacillus casei. Surface localization of the fusion protein was verified by cellular fractionation analyses, immunofluorescence microscopy, and flow cytometry. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G (IgG) and mucosal IgA, as demonstrated by enzyme-linked immunosorbent assays using S protein peptides. More importantly, these antibodies exhibited potent neutralizing activities against severe acute respiratory syndrome (SARS) pseudoviruses. Orally immunized mice mounted a greater neutralizing-antibody response than those immunized intranasally. Three new neutralizing epitopes were identified on the S protein using a peptide neutralization interference assay (residues 291 to 308, 520 to 529, and 564 to 581). These results indicate that mucosal immunization with recombinant L. casei expressing SARS-associated coronavirus S protein on its surface provides an effective means for eliciting protective immune response against the virus.

Novel antibody/gold nanoparticle/magnetic nanoparticle nanocomposites for immunomagnetic separation and rapid colorimetric detection of Staphylococcus aureus in milk

We demonstrated the new antibody/gold nanoparticle/magnetic nanoparticle nanocomposites (antibody/AuNP/MNPs) and their application in the detection of the foodborne pathogen, Staphylococcus aureus (S. aureus), in milk. The nanocomposites were synthesized by coating the MNPs with bovine serum albumin (BSA) then adsorbing the AuNPs and anti-S. aureus antibodies on their surface. Using the completed immunomagnetic nanostructures, S. aureus inoculated in the milk sample was captured and isolated from the medium using the permanent magnet. The nanoparticle-bound cells as well as the unbound cells in the supernatant were enumerated via surface plating to evaluate the target binding capacity of the nanocomposites. The capture efficiencies of the antibody/AuNP/MNPs were 96% and 78% for S. aureus in PBS and the milk sample respectively, which were significantly higher than those of the antibody-coupled MNPs without any AuNP. The captured cells were also applied to the selective filtration system to produce color signals that were used for the detection of the target pathogen. During the filtration, the cells bound to the antibody/AuNP/MNPs remained on the surface of the membrane filter while unbound nanoparticles passed through the uniform pores of the membrane. After the gold enhancement, the cells-particles complex resting on the membrane surface rendered a visible color, and the signal intensity became higher as the target cell concentration increased. The detection limits of this colorimetric sensor were 1.5×10(3) and 1.5×10(5)CFU for S. aureus in PBS and the milk sample respectively. This sensing mechanism also had the high specificity for S. aureus over the other pathogens such as Escherichia coli, Listeria monocytogenes, and Salmonella enterica. The assay required only 40min to obtain the results. With the use of the appropriate antibodies, our immunomagnetic nanocomposites-based detection strategy can provide an easy, convenient, and rapid sensing method for a wide range of pathogens.

Highly Pathogenic Avian Influenza Virus (H5N1) in Domestic Poultry and Relationship with Migratory Birds, South Korea

During the 2006–2007 winter season in South Korea, several outbreaks of highly pathogenic avian influenza virus (H5N1) were confirmed among domestic poultry and in migratory bird habitats. Phylogenetic analysis showed that all isolates were closely related and that all belong to the A/bar-headed goose/Qinghai/5/2005–like lineage rather than the A/chicken/Korea/ES/2003–like lineage.

α-Melanocyte-stimulating Hormone Inhibits Lipopolysaccharide-induced Tumor Necrosis Factor-α Production in Leukocytes by Modulating Protein Kinase A, p38 Kinase, and Nuclear Factor κB Signaling Pathways

The neuropeptide α-melanocyte-stimulating hormone (α-MSH) inhibits inflammation by down-regulating the expression of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) in leukocytes via stimulation of α-MSH cell surface receptors. However, the signaling mechanism of α-MSH action has not yet been clearly elucidated. Here, we have investigated signaling pathways by which α-MSH inhibits lipopolysaccharide (LPS)-induced TNF-α production in leukocytes such as THP-1 cells. We focused on the possible roles of protein kinase A (PKA), p38 kinase, and nuclear factor κB (NFκB) signaling. In THP-1 cells, LPS is known to activate p38 kinase, which in turn activates NFκB to induce TNF-α production. We found that pretreatment of cells with α-MSH blocked LPS-induced p38 kinase and NFκB activation as well as TNF-α production. This response was proportional to α-MSH receptor expression levels, and addition of an α-MSH receptor antagonist abolished the inhibitory effects. In addition, α-MSH treatment activated PKA, and PKA inhibition abrogated the inhibitory effects of α-MSH on p38 kinase activation, NFκB activation, and TNF-α production. Taken together, our results indicate that stimulation of PKA by α-MSH causes inhibition of LPS-induced activation of p38 kinase and NFκB to block TNF-α production.

Isolation and Genetic Characterization of H5N2 Influenza Viruses from Pigs in Korea

ABSTRACT Due to dual susceptibility to both human and avian influenza A viruses, pigs are believed to be effective intermediate hosts for the spread and production of new viruses with pandemic potential. In early 2008, two swine H5N2 viruses were isolated from our routine swine surveillance in Korea. The sequencing and phylogenetic analysis of surface proteins revealed that the Sw/Korea/C12/08 and Sw/Korea/C13/08 viruses were derived from avian influenza viruses of the Eurasian lineage. However, although the Sw/Korea/C12/08 isolate is an entirely avian-like virus, the Sw/Korea/C13/08 isolate is an avian-swine-like reassortant with the PB2, PA, NP, and M genes coming from a 2006 Korean swine H3N1-like virus. The molecular characterization of the two viruses indicated an absence of significant mutations that could be associated with virulence or binding affinity. However, animal experiments showed that the reassortant Sw/Korea/C13/08 virus was more adapted and was more readily transmitted than the purely avian-like virus in a swine experimental model but not in ferrets. Furthermore, seroprevalence in swine sera from 2006 to 2008 suggested that avian H5 viruses have been infecting swine since 2006. Although there are no known potential clinical implications of the avian-swine reassortant virus for pathogenicity in pigs or other species, including humans, at present, the efficient transmissibility of the swine-adapted H5N2 virus could facilitate virus spread and could be a potential model for pandemic, highly pathogenic avian influenza (e.g., H5N1 and H7N7) virus outbreaks or a pandemic strain itself.

Oral Administration of High Molecular Mass Poly-γ-Glutamate Induces NK Cell-Mediated Antitumor Immunity

We analyzed the in vivo tumor regression activity of high molecular mass poly-γ-glutamate (γ-PGA) from Bacillus subtilis sups. chungkookjang. C57BL/6 mice were orally administered 10-, 100-, or 2000-kDa γ-PGA or β-glucan (positive control), and antitumor immunity was examined. Our results revealed higher levels of NK cell-mediated cytotoxicity and IFN-γ secretion in mice treated with higher molecular mass γ-PGA (2000 kDa) vs those treated with lower molecular mass γ-PGA (10 or 100 kDa) or β-glucan. We then examined the effect of oral administration of 10- or 2000-kDa γ-PGA on protection against B16 tumor challenge in C57BL/6 mice. Mice receiving high molecular mass γ-PGA (2000 kDa) showed significantly smaller tumor sizes following challenge with the MHC class I-down-regulated tumor cell lines, B16 and TC-1 P3 (A15), but not with TC-1 cells, which have normal MHC class I expression. Lastly, we found that γ-PGA-induced antitumor effect was decreased by in vivo depletion of NK cells using mAb PK136 or anti-asialo GM1 Ab, and that was completely blocked in NK cell-deficient B6 beige mice or IFN-γ knockout mice. Taken together, we demonstrated that oral administration of high molecular mass γ-PGA (2000 kDa) generated significant NK cell-mediated antitumor activity in mice bearing MHC class I-deficient tumors.

Sublingual Immunization with M2-Based Vaccine Induces Broad Protective Immunity against Influenza

Background The ectodomain of matrix protein 2 (M2e) of influenza A virus is a rationale target antigen candidate for the development of a universal vaccine against influenza as M2e undergoes little sequence variation amongst human influenza A strains. Vaccine-induced M2e-specific antibodies (Abs) have been shown to display significant cross-protective activity in animal models. M2e-based vaccine constructs have been shown to be more protective when administered by the intranasal (i.n.) route than after parenteral injection. However, i.n. administration of vaccines poses rare but serious safety issues associated with retrograde passage of inhaled antigens and adjuvants through the olfactory epithelium. In this study, we examined whether the sublingual (s.l.) route could serve as a safe and effective alternative mucosal delivery route for administering a prototype M2e-based vaccine. The mechanism whereby s.l. immunization with M2e vaccine candidate induces broad protection against infection with different influenza virus subtypes was explored. Methods and Results A recombinant M2 protein with three tandem copies of the M2e (3M2eC) was expressed in Escherichia coli. Parenteral immunizations of mice with 3M2eC induced high levels of M2e-specific serum Abs but failed to provide complete protection against lethal challenge with influenza virus. In contrast, s.l. immunization with 3M2eC was superior for inducing protection in mice. In the latter animals, protection was associated with specific Ab responses in the lungs. Conclusions The results demonstrate that s.l. immunization with 3M2eC vaccine induced airway mucosal immune responses along with broad cross-protective immunity to influenza. These findings may contribute to the understanding of the M2-based vaccine approach to control epidemic and pandemic influenza infections.

Interspecies transmission of the canine influenza H3N2 virus to domestic cats in South Korea, 2010

In the past 4 years, incidences of endemic or epidemic respiratory diseases associated with canine influenza H3N2 virus in Asian dogs have been reported in countries such as South Korea and China. Canine species were considered to be the new natural hosts for this virus. However, at the beginning of 2010, influenza-like respiratory signs, such as dyspnoea, were also observed among cats as well as in dogs in an animal shelter located in Seoul, South Korea. The affected cats showed 100 % morbidity and 40 % mortality. We were able to isolate a virus from a lung specimen of a dead cat, which had suffered from the respiratory disease, in embryonated-chicken eggs. The eight viral genes isolated were almost identical to those of the canine influenza H3N2 virus, suggesting interspecies transmission of canine influenza H3N2 virus to the cat. Moreover, three domestic cats infected with intranasal canine/Korea/GCVP01/07 (H3N2) all showed elevated rectal temperatures, nasal virus shedding and severe pulmonary lesions, such as suppurative bronchopneumonia. Our study shows, for the first time, that cats are susceptible to canine influenza H3N2 infection, suggesting that cats may play an intermediate host role in transmitting the H3N2 virus among feline and canine species, which could lead to the endemic establishment of the virus in companion animals. Such a scenario raises a public health concern, as the possibility of the emergence of new recombinant feline or canine influenza viruses in companion animals with the potential to act as a zoonotic infection cannot be excluded.

Synthesis and high performance of magnetofluorescent polyelectrolyte nanocomposites as MR/near-infrared multimodal cellular imaging nanoprobes.

Here, we describe an easy but robust chemical strategy to synthesize high-performance magnetic resonance (MR)/near-infrared (NIR) multimodal imaging nanoprobes. Poly(γ-glutamic acid) was used for the convenient phase transfer of MnFe(2)O(4) nanoparticles dispersed in organic solvents into aqueous solutions and facilitated further ionic gelation with poly(l-lysine). During the gelation process, MnFe(2)O(4) nanoparticulate satellites were encapsulated in the ionic nanocomplex, which induced synergistic magnetism and resulted in huge T(2) relaxivity (r(2)). The positively charged outer surfaces were assembled with other negatively charged NIR emitting fluorescent nanocrystals and enabled the highly efficient delivery of the magnetofluorescent polyelectrolyte nanocomposites (MagFL-PEN) into cancer cells. The enhancement of negative contrast of MagFL-PEN at 2 μg/mL concentration was similar to that of Resovist at 20 μg/mL concentration. The NIR fluorescence microscopy images of the MagFL-PEN-labeled cells even at 12.5 pM were able to be clearly observed. The labeling efficiency of MagFL-PEN was approximately 65-fold higher compared to that of the commercialized fluorescent nanocrystals, only after 3 h incubation period, even at the test concentration (100 pM). Due to the high-performance capabilities both in materials properties and cell labeling efficiency, the MagFL-PEN is expected to be used as a highly efficient MR/NIR dual-modality imaging nanoprobe in the detection of cancer cells and monitoring of therapeutic cells in vivo.

Oral administration of human papillomavirus type 16 E7 displayed on Lactobacillus casei induces E7‐specific antitumor effects in C57/BL6 mice

The mounting of a specific immune response against the human papillomavirus type 16 E7 protein (HPV16 E7) is important for eradication of HPV16 E7‐expressing cancer cells from the cervical mucosa. To induce a mucosal immune response by oral delivery of the E7 antigen, we expressed the HPV16 E7 antigen on the surface of Lactobacillus casei by employing a novel display system in which the poly‐γ‐glutamic acid (γ‐PGA) synthetase complex A (PgsA) from Bacillus subtilis (chungkookjang) was used as an anchoring motif. After surface expression of the HPV16 E7 protein was confirmed by Western blot, flow cytometry and immunofluorescence microscopy, mice were orally inoculated with L. casei‐PgsA‐E7. E7‐specific serum IgG and mucosal IgA productions were enhanced after oral administration and significantly enhanced after boosting. Systemic and local cellular immunities were significantly increased after boosting, as shown by increased counts of lymphocytes (SI = 9.7 ± 1.8) and IFN‐γ secreting cells [510 ± 86 spot‐forming cells/106cells] among splenocytes and increased IFN‐γ in supernatants of vaginal lymphocytes. Furthermore, in an E7‐based mouse tumor model, animals receiving orally administered L. casei‐PgsA‐E7 showed reduced tumor size and increased survival rate versus mice receiving control (L. casei‐PgsA) immunization. These results collectively indicate that the oral administration of E7 displayed on lactobacillus induces cellular immunity and antitumor effects in mice.