Protection from hemolytic uremic syndrome by eyedrop vaccination with modified enterohemorrhagic E. coli outer membrane vesicles

We investigated whether eyedrop vaccination using modified outer membrane vesicles (mOMVs) is effective for protecting against hemolytic uremic syndrome (HUS) caused by enterohemorrhagic E. coli (EHEC) O157:H7 infection. Modified OMVs and waaJ-mOMVs were prepared from cultures of MsbB- and Shiga toxin A subunit (STxA)-deficient EHEC O157:H7 bacteria with or without an additional waaJ mutation. BALB/c mice were immunized by eyedrop mOMVs, waaJ-mOMVs, and mOMVs plus polymyxin B (PMB). Mice were boosted at 2 weeks, and challenged peritoneally with wild-type OMVs (wtOMVs) at 4 weeks. As parameters for evaluation of the OMV-mediated immune protection, serum and mucosal immunoglobulins, body weight change and blood urea nitrogen (BUN)/Creatinin (Cr) were tested, as well as histopathology of renal tissue. In order to confirm the safety of mOMVs for eyedrop use, body weight and ocular histopathological changes were monitored in mice. Modified OMVs having penta-acylated lipid A moiety did not contain STxA subunit proteins but retained non-toxic Shiga toxin B (STxB) subunit. Removal of the polymeric O-antigen of O157 LPS was confirmed in waaJ-mOMVs. The mice group vaccinated with mOMVs elicited greater humoral and mucosal immune responses than did the waaJ-mOMVs and PBS-treated groups. Eyedrop vaccination of mOMVs plus PMB reduced the level of humoral and mucosal immune responses, suggesting that intact O157 LPS antigen can be a critical component for enhancing the immunogenicity of the mOMVs. After challenge, mice vaccinated with mOMVs were protected from a lethal dose of wtOMVs administered intraperitoneally, conversely mice in the PBS control group were not. Collectively, for the first time, EHEC O157-derived mOMV eyedrop vaccine was experimentally evaluated as an efficient and safe means of vaccine development against EHEC O157:H7 infection-associated HUS.ope

Inactivated Eyedrop Influenza Vaccine Adjuvanted with Poly(I:C) Is Safe and Effective for Inducing Protective Systemic and Mucosal Immunity

The eye route has been evaluated as an efficient vaccine delivery routes. However, in order to induce sufficient antibody production with inactivated vaccine, testing of the safety and efficacy of the use of inactivated antigen plus adjuvant is needed. Here, we assessed various types of adjuvants in eyedrop as an anti-influenza serum and mucosal Ab production-enhancer in BALB/c mice. Among the adjuvants, poly (I:C) showed as much enhancement in antigen-specific serum IgG and mucosal IgA antibody production as cholera toxin (CT) after vaccinations with trivalent hemagglutinin-subunits or split H1N1 vaccine antigen in mice. Vaccination with split H1N1 eyedrop vaccine antigen plus poly(I:C) showed a similar or slightly lower efficacy in inducing antibody production than intranasal vaccination; the eyedrop vaccine-induced immunity was enough to protect mice from lethal homologous influenza A/California/04/09 (H1N1) virus challenge. Additionally, ocular inoculation with poly(I:C) plus vaccine antigen generated no signs of inflammation within 24 hours: no increases in the mRNA expression levels of inflammatory cytokines nor in the infiltration of mononuclear cells to administration sites. In contrast, CT administration induced increased expression of IL-6 cytokine mRNA and mononuclear cell infiltration in the conjunctiva within 24 hours of vaccination. Moreover, inoculated visualizing materials by eyedrop did not contaminate the surface of the olfactory bulb in mice; meanwhile, intranasally administered materials defiled the surface of the brain. On the basis of these findings, we propose that the use of eyedrop inactivated influenza vaccine plus poly(I:C) is a safe and effective mucosal vaccine strategy for inducing protective anti-influenza immunity.ope

CD11b+ 수지상세포에 의해 매개되는 점안형 불활성화 백신에 대한 면역반응 유발

Dept. of Medical Science/박사The eye route has been evaluated as an efficient vaccine delivery routes. However, in order to induce sufficient antibody production with inactivated vaccine, testing of the safety and efficacy of the use of inactivated Ag plus adjuvant is needed. Here, I assessed various types of adjuvants in eyedrop as an anti-influenza serum and mucosal Ab production-enhancer in BALB/c mice. Among the adjuvants, poly (I:C) showed as much enhancement in Ag-specific serum IgG and mucosal IgA antibody production as cholera toxin (CT) after vaccinations with trivalent hemagglutinin-subunits or split H1N1 vaccine Ag in mice. Vaccination with split H1N1 eyedrop vaccine (EDV) Ag plus poly(I:C) showed a similar or slightly lower efficacy in inducing antibody production than intranasal vaccination; the EDV-induced immunity was enough to protect mice from lethal homologous influenza A/California/04/09 (H1N1) virus challenge. Additionally, ocular inoculation with poly(I:C) plus vaccine Ag generated no signs of inflammation within 24 hours: no increases in the mRNA expression levels of inflammatory cytokines nor in the infiltration of mononuclear cells to administration sites. In contrast, CT administration induced increased expression of IL-6 cytokine mRNA and mononuclear cell infiltration in the conjunctiva within 24 hours of vaccination. Moreover, inoculated visualizing materials by eyedrop did not contaminate the surface of the olfactory bulb in mice; meanwhile, intranasally administered materials defiled the surface of the brain. On the basis of these findings, I propose that the use of inactivated influenza EDV plus poly(I:C) is a safe and effective mucosal vaccine strategy for inducing protective anti-influenza immunity. Although the efficacy of inactivated EDV was confirmed, the type of antigen presenting cells (APCs) that mediates antigen-specific immune induction has not been reported. Moreover, how the EDV is delivered into the draining lymph nodes (dLN), which are mandibular lymph nodes (MdLN) and superficial parotid lymph nodes (SPLN), is not clarified. In here, I showed that the delivery of proteins or fluorescent beads into the dLN administered by eyedrop or subconjunctival (SCJ) injection is not dependent on the migration of dendritic cells (DCs) or the activation of DCs by TLR stimulation. Instead, the particulates were delivered by flow of lymphatic drainages into the dLN. Among two comprising parts of the dLN, cells in MdLN showed higher levels of percentages of PE-beads+ than SPLN. In MdLN, CD11b+ DCs were in significantly higher percentages of phycoerythrin (PE)-beads+ than other subsets of DCs do in both resident and migratory DCs. In CD11b knockout mice, the levels of antigen-specific serum IgG or mucosal IgA production were significantly decreased. Thus, it is expected that the strategy targeting resident CD11b+ DCs in MdLN utilizing lymphatic drainage can strengthen the development of effective EDV. 약독화된 생백신을 마우스에 점안으로 접종한 점안백신의 효능이 발표된 이후, 점안백신은 전신 면역과 점막 면역을 동시에 일으키는 점막 백신으로서 새로운 형태의 점막 백신으로 제시되고 있다. 이에 따라, 본 연구에서 생백신보다 안전한 불활성화된 인플루엔자 백신 및 기타 상용화된 백신들의 효능을 점안 접종법에서 확인하였으며, 인플루엔자 백신 연구에 주로 사용되는 페렛에서 점안백신의 효능을 확인하였다. 또한 마우스에서 점안백신 항원을 전달하는 항원 제시세포의 종류와 그 아형을 동정하였다. 82 불활성화 인플루엔자 백신 항원을 점안 백신으로 사용하기 위하여 적합한 아주번트를 일 차적으로 확인해본 결과, 효과는 가장 좋지만 독성이 있는 콜레라톡신을 대체할 아주번트로 poly(I:C)가 적합함을 확인하였다. Poly(I:C)를 아주번트로 사용할 경우 서브 유닛 및 분할 인플루엔자 백신 항원을 점안으로 접종할 때 혈청 및 점막액에서 항원특이 항체가 유의하게 증가하였다. H1N1 바이러스 분할 백신을 poly(I:C)와 함께 점안으로 접종할 경우 백신에 접종된 마우스들은 치명적인 인플루엔자 바이러스 감염에 보호되었으며, 백신 접종 부위인 눈에서 염증반응이 유발되지 않고 백신 물질이 중추신경계로 흡수되지 않았다. 한 편, 페렛에서는 H1N1, H1N2, H3N2의 세 가지 다른 약독화 인플루엔자 생바이러스를 백신항원으로 점안접종 한 후 감염성이 있는 인플루엔자 바이러스를 감염시켰다. 그 결과 점안백신을 접종 받은 페렛들에서 대조군에 비해 바이러스 중화 항체가 유의하게 증가하였으며 체중감소와 체온상승이 일어나지 않았다. 또한 감염된 바이러스는 5일 안에 폐에서 모두 제거되었으며, 마우스와 마찬가지로 눈 조직에서의 염증반응이 일어나지 않았다. 따라서 점안으로 불활성화 인플루엔자 백신을 poly(I:C)와 함께 접종하는 것이 새로운 안전하고 효과적인 점막 인플루엔자 백신 접종법이 될 수 있으며, 페렛 모델에서 확인한 바를 바탕으로 임상 단계에서의 실험이 가능할 것으로 기대된다. 또한, 기존의 근육주사로 접종되고 있는 상용화된 백신들을 점안...ope

Effect of voriconazole and ultraviolet - A combination therapy compared to voriconazole single treatment on fusarium solani fungal keratitis

Purpose: To demonstrate that ultraviolet-A (UV-A) and voriconazole combination therapy is more effective than voriconazole single treatment for fungal keratitis. Methods: The in vitro UV-A (375 nm) fungicidal effect was evaluated on Fusarium solani solutions. Each fungal solution was irradiated with different UV-A irradiation doses. Also, a fungal solution containing voriconazole was also irradiated with UV-A. The in vivo therapeutic effect of UV-A and voriconazole treatment was studied in a rabbit keratitis model. Fungi were injected intrastromally into the cornea of 16 rabbits. Each treatment was initiated 3 days after fungal injection and continued up to 8 days for the following groups: Group 1, control; Group 2, treated with UV-A once a day; Group 3, treated with voriconazole 3 times a day; Group 4, treated with voriconazole 3 times a day and UV-A once a day. On the last day, the sclera–cornea buttons were extracted and microbiological and histological evaluations were performed. Results: The colony-forming units (CFUs) of fungal solutions in culture significantly decreased with UV-A irradiation. The CFUs of fungal solutions containing voriconazole also decreased with UV-A irradiation. In vivo, clinical scores of Group 3 (P=0.03) and Group 4 (P=0.02) 5 days after treatment were significantly lower compared to that of Group 1. The clinical score of Group 4 (P=0.03) 5 days after treatment was significantly lower compared to that of Group 3. The histopathological scores 5 days after treatment were significantly lower in Group 4 compared to those of Group 1 (P<0.01) and Group 3 (P=0.02). Based on our CFU analysis, only Group 4 showed significantly lower CFUs compared to Group 1 (P=0.04). Conclusions: UV-A and voriconazole combination treatment could be a safe and effective alternative to voriconazole single treatment for fungal keratitis.ope