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Ong Y.T.,University of Missouri | Kirby K.A.,University of Missouri | Hachiya A.,University of Missouri | Hachiya A.,Clinical Center | And 7 more authors.
Cellular and Molecular Biology | Year: 2012

KD-247 is a humanized monoclonal antibody that targets the third hypervariable (V3) loop of gp120. It can efficiently neutralize a broad panel of clade B, but not non-clade B, HIV-1 isolates. To overcome this limitation, we are seeking to prepare genetically-engineered single-chain variable fragments (scFvs) of KD-247 that will have broader neutralizing activity against both clade B and non-clade B HIV-1 isolates. Initial attempts of optimizing the expression of KD-247 scFv have resulted in the formation of insoluble protein. Therefore, we have established purification protocols to recover, purify, and refold the KD-247 scFv from inclusion bodies. The protocol involved step-wise refolding of denatured scFv by dilution, dialysis, and on-column nickel-affinity purification. Monomeric scFv was further purified by size-exclusion chromatography. Using far UV circular dichroism (CD) spectroscopy we confirmed the expected beta-sheet profile of the refolded KD-247 scFv. Importantly, the refolded KD-247 scFv showed neutralizing activity against replication-competent HIV-1 BaL and JR-FL Env pseudotyped HIV-1, at potency comparable to that of the native full-size KD-247 antibody. Ongoing studies focus on the application of this system in generating KD-247 scFv variants with the ability to neutralize clade B and non-clade B HIV-1 isolates. © 2012.

Chisada S.-I.,Kyushu University | Chisada S.-I.,Japan National Research Institute of Fisheries And Environment of Inland Sea | Shimizu K.,Kyushu University | Shimizu K.,The Chemo Sero Therapeutic Research Institute Kaketsuken | And 5 more authors.
FEMS Microbiology Letters | Year: 2013

Vibrios, distributed in marine and brackish environments, can cause vibriosis in fish and shellfish under appropriate conditions. Previously, we clarified by thin-layer chromatography (TLC) overlay assay that 35S-labeled Vibrio trachuri adhered to GM4 isolated from red sea bream intestine. However, whether GM4 actually functions on epithelial cells as an attachment site for vibrios still remains to be uncovered. We found that six isolates, classified as V. harveyi, V. campbellii, and V. splendidus, from intestinal microflora of red sea bream adhered to GM4 but not galactosylceramide (GalCer) by TLC-overlay assay. Tissue-overlay assays revealed that V. harveyi labeled with green fluorescent protein (GFP) adhered to epithelial cells of red sea bream intestine where GM4 and GalCer were found to be distributed on the top layer of actin filaments by immunohistochemical analysis using corresponding antibodies. The number of adhering vibrios was diminished by pretreatment with anti-GM4 antibody, but not anti-GalCer antibody. These results clearly indicate that vibrios adhere to epithelial cells of red sea bream intestine utilizing GM4 as an attachment site. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.

Niimi N.,Tokyo Metropolitan Institute for Neuroscience | Kohyama K.,Tokyo Metropolitan Institute for Neuroscience | Kamei S.,The Chemo Sero Therapeutic Research Institute Kaketsuken | Matsumoto Y.,Tokyo Metropolitan Institute for Neuroscience
Neuropathology | Year: 2011

Although intravenous immunoglobulin (IVIG) has been reported to improve the status of expanded disability status scale (EDSS) of multiple sclerosis (MS) patients and reduce the annual relapse rate, some studies did not find its beneficial effects. In the present study, using an animal model for MS, we found that prophylactic, but not therapeutic, treatment successfully suppressed the disease development. During the search for factors involved in the disease suppression by IVIG, we obtained evidence suggesting that IVIG exerts its function, at least in part, by suppressing activation of matrix metalloproteinases (MMP)-2 and -9. Gelatin zymography revealed that gelatinase activities were suppressed by IVIG treatment in the spinal cord, but not in plasma. This finding raises the possibility that IVIG blocks MMP activities at the interface between the blood stream and CNS. With in situ zymography, we also observed that gelatinase activities were expressed mainly in astrocytes in the inflamed spinal cord of control rats and that this expression was attenuated by the treatment. These findings provide useful information to set optimal conditions for IVIG treatment of MS and to obtain more beneficial effects. © 2010 Japanese Society of Neuropathology.

Nishiyama Y.,Health Care Center | Fujii T.,Health Care Center | Kanatani Y.,Japan National Institute of Public Health | Shinmura Y.,The Chemo Sero Therapeutic Research Institute Kaketsuken | And 2 more authors.
Vaccine | Year: 2015

Background: In Japan, production of smallpox vaccine LC16m8 (named LC16-KAKETSUKEN) was restarted and was determined to be maintained as a national stockpile in March 2002. Objective: To conduct a post-marketing surveillance study of the vaccination of freeze-dried live attenuated smallpox vaccine prepared in cell culture LC16-KAKETSUKEN using attenuated vaccinia strain LC16m8. The study complied with Good Clinical Practice, focusing on a comparison between primary vaccinees and re-vaccinees. Method: 268 personnel (261 males and 7 females) of the Japan Ground Self-Defense Force were inoculated with LC16-KAKETSUKEN and thereafter adverse events and efficacy were evaluated. Results: Among 268 vaccinee participants, the following vaccinees showed adverse events, none serious: 53 of 196 primary vaccinees (without previous smallpox vaccination), 4 of 71 re-vaccinees (with previous smallpox vaccination) and 1 vaccinee with unknown previous vaccination history. A breakdown of adverse events observed in this study (total 268 vaccinees) showed the following minor or mild adverse events: 52 (19.4%) swelling of axillary lymph node, 4 (1.5%) fever, 2 (0.7%) fatigue, 1 (0.4%) of rash, 14 (5.2%) erythema at the inoculation site, 1 (0.4%) swelling at the inoculation site and 1 (0.4%) autoinoculation. The incidence of adverse events for primary vaccinees (53/196; 27.0%) was significantly higher than for re-vaccinees (4/71; 5.6%). However, the proportion of vaccine take was significantly higher for primary vaccinees (185/196; 94.4%) than for re-vaccinees (58/71; 81.7%). Although the proportion of vaccine take of re-vaccinees was significantly lower than for primary vaccinees due to preexisting immunity by previous vaccination, no significant difference was found in neutralizing antibody titers between primary vaccinees and re-vaccinees at 1, 4 and 7 months after LC16-KAKETSUKEN vaccination. Conclusion: The present post-marketing surveillance study compliant with Good Clinical Practice demonstrated the efficacy and safety of the smallpox vaccine LC16-KAKETSUKEN in an adult population. LC16-KAKETSUKEN is the sole currently available licensed smallpox vaccine for both adult and pediatric populations. © 2015 The Authors.

Naruse T.,The Chemo Sero Therapeutic Research Institute Kaketsuken | Fukuda T.,The Chemo Sero Therapeutic Research Institute Kaketsuken | Tanabe T.,The Chemo Sero Therapeutic Research Institute Kaketsuken | Ichikawa M.,The Chemo Sero Therapeutic Research Institute Kaketsuken | And 5 more authors.
Vaccine | Year: 2015

Background: We conducted a phase I clinical trial of a cell culture-derived AS03-adjuvanted influenza vaccine containing HA antigen (A/Indonesia/05/2005(H5N1)/PR8-IBCDC-RG2) derived from EB66 cells (KD-295). Methods: Healthy male adult volunteers (20-40 years old, N= 60) enrolled in the study were divided into 3 groups, the MA group (3.8. μg of HA + AS03), HA group (7.5. μg of HA + AS03), and 1/2 MA group (half the volume of the MA group), and received KD-295 intramuscularly twice with a 21-day interval. After administration of KD-295, adverse events, clinical laboratory parameters, and immune response to the vaccine strain and heterologous virus strains were evaluated. Results: No severe adverse events leading to discontinuation of vaccine administration occurred. The vaccine was well-tolerated. There was no dose dependency in the rate, timing, or duration of the adverse events. Immunogenicity of the vaccines was evaluated by HI (hemagglutination inhibition) assay, which confirmed that the antibody response to the vaccine strain and heterologous strain in all groups met the three criteria for immunogenicity described in the Japanese guidelines for development of a pandemic prototype vaccine. We also measured the neutralizing antibody titers against several virus strains, and confirmed a significant rise in antibody levels to both the vaccine strain and heterologous strains. Conclusion: The EB66-derived H5N1 influenza vaccine adjuvanted with AS03 elicited a broad cross-reactive antibody response among H5N1 strains with acceptable reactogenicity. Therefore, KD-295 can be considered a useful pandemic and pre-pandemic influenza vaccine candidate. © 2015 Elsevier Ltd.

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