Protective efficacy of a bivalent H5 influenza vaccine candidate against both clades 2.3.2.1 and 2.3.4.4 high pathogenic avian influenza viruses in SPF chickens.

Worldwide, high pathogenic avian influenza viruses belonging to clades 2.3.4.4 and 2.3.2.1 have been circulating in both poultry and wild birds. Since 2018, Korea has built a national antigen bank to ensure preparedness in an emergency. In this study, we developed a bivalent vaccine candidate containing antigens derived from two reassortant KA435/2.3.2.1d and H35/2.3.4.4b strains for Korean national antigen bank. We evaluated its immunogenicity and protective efficacy in specific pathogen free chickens. The two vaccine strains, rgKA435-H9N2 PB2/2.3.2.1d and rgH35/2.3.4.4b, both of which were generated successfully by reverse genetics, were highly immunogenic (titres of haemagglutination inhibition: 8.3 and 8.4 log2, respectively) and showed good protective efficacy (100 and 147 50% protective dose, respectively) against lethal challenge with wild-type virus when delivered as a 1:1 mixture. Notably, the vaccine provided complete protection against viral shedding at a full dose (512 HAU) and a 1/10 dose (51.2 HAU), with no clinical signs, after challenge with H35/2.3.4.4b. The bivalent vaccine developed in this study may reduce the cost of vaccine production and could be used as a H5 subtype avian influenza vaccine candidate against two clades simultaneously.

Protection of SPF Chickens by H9N2 Y439 and G1 Lineage Vaccine against Homologous and Heterologous Viruses.

Prior to the identification of low pathogenic avian influenza H9N2 viruses belonging to the Y280 lineage in 2020, Y439 lineage viruses had been circulating in the Republic of Korea since 1996. Here, we developed a whole inactivated vaccine (vac564) by multiple passage of Y439 lineage viruses and then evaluated immunogenicity and protective efficacy in specific-pathogen-free chickens. We found that LBM564 could be produced at high yield in eggs (108.4EID50/0.1 mL; 1024 hemagglutinin units) and was immunogenic (8.0 ± 1.2 log2) in chickens. The vaccine showed 100% inhibition of virus in the cecal tonsil with no viral shedding detected in either oropharyngeal or cloacal swabs after challenge with homologous virus. However, it did not induce effective protection against challenge with heterologous virus. An imported commercial G1 lineage vaccine inhibited viral replication against Y280 and Y439 lineage viruses in major tissues, although viral shedding in oropharyngeal and cloacal swabs was observed up until 5 dpi after exposure to both challenge viruses. These results suggest that a single vaccination with vac564 could elicit immune responses, showing it to be capable of protecting chickens against the Y439 lineage virus. Thus, our results suggest the need to prepare suitable vaccines for use against newly emerging and re-emerging H9N2 viruses.

Updating the National Antigen Bank in Korea: Protective Efficacy of Synthetic Vaccine Candidates against H5Nx Highly Pathogenic Avian Influenza Viruses Belonging to Clades 2.3.2.1 and 2.3.4.4.

Since 2018, Korea has been building an avian influenza (AI) national antigen bank for emergency preparedness; this antigen bank is updated every 2 years. To update the vaccine strains in the antigen bank, we used reverse genetics technology to develop two vaccine candidates against avian influenza strains belonging to clades 2.3.2.1d and 2.3.4.4h, and then evaluated their immunogenicity and protective efficacy in SPF chickens challenged with H5 viruses. The two vaccine candidates, named rgCA2/2.3.2.1d and rgES3/2.3.4.4h, were highly immunogenic, with hemagglutination inhibition (HI) titers of 8.2−9.3 log2 against the vaccine strain, and 7.1−7.3 log2 against the lethal challenge viruses (in which the HA genes shared 97% and 95.4% homology with that of rgCA2/2.3.2.1d and rgES3/2.3.4.4h, respectively). A full dose of each vaccine candidate provided 100% protection against the challenge viruses, with a reduction in clinical symptoms and virus shedding. A 1/10 dose provided similar levels of protection, whereas a 1/100 dose resulted in mortality and virus shedding by 7 dpi. Moreover, immunity induced by the two vaccines was long lasting, with HI titers of >7 log2 against the vaccine strain remaining after 6 months. Thus, the two vaccine candidates show protective efficacy and can be used to update the AI national antigen bank.

Immunogenicity of an inactivated rabies vaccine for animals derived from the recombinant ERAGS strain.

PURPOSE: The aims of the present study were to evaluate the immunogenicity of an inactivated rabies vaccine based on the ERAGS strain. MATERIALS AND METHODS: The ERAGS virus propagated in Vero cells was inactivated with 3 mM binary ethylenimine for 8 hours. Three types of inactivated rabies vaccines were prepared to determine the minimum vaccine virus titers. Four further types of inactivated rabies vaccines were prepared by blending inactivated ERAGS with four different adjuvants; each vaccine was injected into mice, guinea pigs, and dogs to identify the optimal adjuvant. The immunogenicity of a Montanide (IMS) gel-adjuvanted vaccine was evaluated in cats, dogs, and cattle. Humoral immune responses were measured via a fluorescent antibody virus neutralization method and a blocking enzyme-linked immunosorbent assay. RESULTS: The minimum virus titer of the inactivated rabies vaccine was over 107.0 50% tissue culture infectious doses (TCID50 values)/mL. Of the four kinds of adjuvants, the IMS gel-adjuvanted vaccine induced the highest mean viral neutralizing antibody (VNA) titers of 6.24 and 2.36 IU/mL in guinea pigs and dogs, respectively, and was thus selected as the vaccine for the target animals. Cats, dogs, and cattle inoculated with the IMS gel-adjuvanted vaccine developed protective VNA titers ranging from 3.5 to 1.2 IU/mL at 4 weeks post-inoculation (WPI). CONCLUSION: Our data indicate that cats, dogs, and cattle inoculated with an inactivated rabies vaccine derived from the ERAGS strain developed protective immune responses that were maintained to 12 WPI.

Engineering Escherichia coli to produce and secrete colicins for rapid and selective biofilm cell killing.

Bacterial biofilms are associated with chronic infectious diseases and are highly resistant to conventional antibiotics. Antimicrobial bacteriocins are alternatives to conventional antibiotics and are characterized by unique cell-killing mechanisms, including pore formation on cell membranes, nuclease activity, and cell wall synthesis inhibition. Here, we used cell-free protein synthesis to rapidly evaluate the anti-biofilm activities of colicins E1, E2, and E3. We found that E2 (with DNase activity) most effectively killed target biofilm cells (i.e., the K361 strain) while leaving non-targeted biofilms intact. We then engineered probiotic Escherichia coli microorganisms with genetic circuits to controllably synthesize and secrete colicin E2, which successfully inhibited biofilms and killed pre-formed indicator biofilms. Our findings suggest that colicins rapidly and selectively kill target biofilm cells in multispecies biofilms and demonstrate the potential of using microorganisms engineered to produce antimicrobial colicin proteins as live therapeutic strategies to treat biofilm-associated infections.

Preparation and evaluation of ß-glucan hydrogel prepared by the radiation technique for drug carrier applications.

ß-Glucan can provide excellent environment to apply to drug carrier due to its immunological and anti-inflammatory effect. Minocycline hydrochloride (MH) has excellent oral bioavailability pharmacological properties. Specifically, MH is effectively absorbed into the gingiva for periodontal disease treatment. In this study, we attempt to develop MH loaded ß-glucan hydrogel for periodontal disease treatment through radiation-crosslinking technique. In addition, MH loaded ß-glucan hydrogels were tested for their cytotoxicity and antibacterial activity. Finally, we conducted an in vivo study to demonstrate the potential to prevent the invasion of bacteria to treat periodontal disease. The gel content and compressive strength of the ß-glucan hydrogels increased as the ß-glucan content and the absorbed dose (up to 7 kGy) increased. For a radiation dose of 7 kGy, the gelation and the compressive strength of a 6 wt% ß-glucan hydrogel were approximately 92% and 270 kPa, respectively. As a drug, MH was consistently released from ß-glucan hydrogels, reaching 80% at approximately 90 min. Furthermore, the MH loaded ß-glucan hydrogels showed no cytotoxicity. The MH loaded ß-glucan hydrogels exhibited good antibacterial activity against Porphyromonas gingivalis. In addition, MH loaded ß-glucan hydrogel demonstrated the potential of a good capability to prevent the invasion of bacteria and to treat wounds.

Preparation and Characterization of Resorbable Bacterial Cellulose Membranes Treated by Electron Beam Irradiation for Guided Bone Regeneration.

Bacterial cellulose (BC) is an excellent biomaterial with many medical applications. In this study, resorbable BC membranes were prepared for guided bone regeneration (GBR) using an irradiation technique for applications in the dental field. Electron beam irradiation (EI) increases biodegradation by severing the glucose bonds of BC. BC membranes irradiated at 100 kGy or 300 kGy were used to determine optimal electron beam doses. Electron beam irradiated BC membranes (EI-BCMs) were evaluated by scanning electron microscopy (SEM), attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, thermal gravimetric analysis (TGA), and using wet tensile strength measurements. In addition, in vitro cell studies were conducted in order to confirm the cytocompatibility of EI-BCMs. Cell viabilities of NIH3T3 cells on 100k and 300k EI-BCMs (100 kGy and 300 kGy irradiated BC membranes) were significantly greater than on NI-BCMs after 3 and 7 days (p < 0.05). Bone regeneration by EI-BCMs and their biodegradabilities were also evaluated using in vivo rat calvarial defect models for 4 and 8 weeks. Histometric results showed 100k EI-BCMs exhibited significantly larger new bone area (NBA; %) than 300k EI-BCMs at 8 weeks after implantation (p < 0.05). Mechanical, chemical, and biological analyses showed EI-BCMs effectively interacted with cells and promoted bone regeneration.

The Efficacy of Electron Beam Irradiated Bacterial Cellulose Membranes as Compared with Collagen Membranes on Guided Bone Regeneration in Peri-Implant Bone Defects.

Bacterial cellulose (BC) is a natural polysaccharide produced by some bacteria, and consists of a linear polymer linked by ß-(1,4) glycosidic bonds. BC has been developed as a material for tissue regeneration purposes. This study was conducted to evaluate the efficacy of resorbable electron beam irradiated BC membranes (EI-BCMs) for guided bone regeneration (GBR). The electron beam irradiation (EI) was introduced to control the biodegradability of BC for dental applications. EI-BCMs had higher porosity than collagen membranes (CMs), and had similar wet tensile strengths to CMs. NIH3T3 cell adhesion and proliferation on EI-BCMs were not significantly different from those on CMs (p > 0.05). Micro-computed tomography (µCT) and histometric analysis in peri-implant dehiscence defects of beagle dogs showed that EI-BCMs were non-significantly different from CMs in terms of new bone area (NBA; %), remaining bone substitute volume (RBA; %) and bone-to-implant contact (BIC; %) (p > 0.05). These results suggest resorbable EI-BCMs can be used as an alternative biomaterial for bone tissue regeneration.

The Effect of Thickness of Resorbable Bacterial Cellulose Membrane on Guided Bone Regeneration.

This study introduces the effect of the thickness of a bacterial cellulose membrane by comparing the bone regeneration effect on rat skulls when using a collagen membrane and different thicknesses of resorbable bacterial cellulose membranes for guided bone regeneration. Barrier membranes of 0.10 mm, 0.15 mm, and 0.20 mm in thickness were made using bacterial cellulose produced as microbial fermentation metabolites. Mechanical strength was investigated, and new bone formation was evaluated through animal experimental studies. Experimental animals were sacrificed after having 2 weeks and 8 weeks of recovery, and specimens were processed for histologic and histomorphometric analyses measuring the area of bone regeneration (%) using an image analysis program. In 2 weeks, bone-like materials and fibrous connective tissues were observed in histologic analysis. In 8 weeks, all experimental groups showed the arrangement of osteoblasts surrounding the supporting body on the margin and center of the bone defect region. However, the amount of new bone formation was significantly higher (p < 0.05) in bacterial cellulose membrane with 0.10 mm in thickness compared to the other experimental groups. Within the limitations of this study, a bacterial cellulose membrane with 0.10 mm thickness induced the most effective bone regeneration.

Chestnut Honey Impregnated Carboxymethyl Cellulose Hydrogel for Diabetic Ulcer Healing.

Honey-based wound dressings have attracted a lot of attention from modern scientists owing to their anti-inflammatory and antibacterial effects without antibiotic resistance. Such dressings also promote moist wound healing, and have been considered natural, abundant, and cheap materials for folk marketing. This study investigated the various behaviors and characteristics of chestnut honey-impregnated carboxymethyl cellulose sodium hydrogel paste (CH⁻CMC) as a therapeutic dressing, such as its moist retention, antibacterial activity for inhibiting the growth of Staphylococcus aureus and Escherichia coli, and the rate of wound healing in db/db mice. The results provide good evidence, suggesting that CH⁻CMC has potential as a competitive candidate for diabetic ulcer wound healing.

The effect of bacterial cellulose membrane compared with collagen membrane on guided bone regeneration.

PURPOSE: This study was to evaluate the effects of bacterial cellulose (BC) membranes as a barrier membrane on guided bone regeneration (GBR) in comparison with those of the resorbable collagen membranes. MATERIALS AND METHODS: BC membranes were fabricated using biomimetic technology. Surface properties were analyzed, Mechanical properties were measured, in vitro cell proliferation test were performed with NIH3T3 cells and in vivo study were performed with rat calvarial defect and histomorphometric analysis was done. The Mann-Whitney U test and the Wilcoxon signed rank test was used (α<.05). RESULTS: BC membrane showed significantly higher mechanical properties such as wet tensile strength than collagen membrane and represented a three-dimensional multilayered structure cross-linked by nano-fibers with 60 % porosity. In vitro study, cell adhesion and proliferation were observed on BC membrane. However, morphology of the cells was found to be less differentiated, and the cell proliferation rate was lower than those of the cells on collagen membrane. In vivo study, the grafted BC membrane did not induce inflammatory response, and maintained adequate space for bone regeneration. An amount of new bone formation in defect region loaded with BC membrane was significantly similar to that of collagen membrane application. CONCLUSION: BC membrane has potential to be used as a barrier membrane, and efficacy of the membrane on GBR is comparable to that of collagen membrane.