Protein engineering of Bacillus acidopullulyticus pullulanase for enhanced thermostability using in silico data driven rational design methods.

Science.gov (United States)

Chen, Ana; Li, Yamei; Nie, Jianqi; McNeil, Brian; Jeffrey, Laura; Yang, Yankun; Bai, Zhonghu

2015-10-01

Thermostability has been considered as a requirement in the starch processing industry to maintain high catalytic activity of pullulanase under high temperatures. Four data driven rational design methods (B-FITTER, proline theory, PoPMuSiC-2.1, and sequence consensus approach) were adopted to identify the key residue potential links with thermostability, and 39 residues of Bacillus acidopullulyticus pullulanase were chosen as mutagenesis targets. Single mutagenesis followed by combined mutagenesis resulted in the best mutant E518I-S662R-Q706P, which exhibited an 11-fold half-life improvement at 60 °C and a 9.5 °C increase in Tm. The optimum temperature of the mutant increased from 60 to 65 °C. Fluorescence spectroscopy results demonstrated that the tertiary structure of the mutant enzyme was more compact than that of the wild-type (WT) enzyme. Structural change analysis revealed that the increase in thermostability was most probably caused by a combination of lower stability free-energy and higher hydrophobicity of E518I, more hydrogen bonds of S662R, and higher rigidity of Q706P compared with the WT. The findings demonstrated the effectiveness of combined data-driven rational design approaches in engineering an industrial enzyme to improve thermostability. Copyright © 2015 Elsevier Inc. All rights reserved.

Purification and Characterization of a Thermostable Lipase from Geobacillus thermodenitrificans IBRL-nra

Directory of Open Access Journals (Sweden)

Anuradha Balan

2012-01-01

Full Text Available Thermostable lipase from Geobacillus thermodenitrificans IBRL-nra was purified and characterized. The production of thermostable lipase from Geobacillus thermodenitrificans IBRL-nra was carried out in a shake-flask system at 65°C in cultivation medium containing; glucose 1.0% (w/v; yeast extract 1.25% (w/v; NaCl 0.45% (w/v olive oil 0.1% (v/v with agitation of 200 rpm for 24 hours. The extracted extracellular crude thermostable lipase was purified to homogeneity by using ultrafiltration, Heparin-affinity chromatography, and Sephadex G-100 gel-filtration chromatography by 34 times with a final yield of 9%. The molecular weight of the purified enzyme was estimated to be 30 kDa after SDS-PAGE analysis. The optimal temperature for thermostable lipase was 65°C and it retained its initial activity for 3 hours. Thermostable lipase activity was highest at pH 7.0 and stable for 16 hours at this pH at 65°C. Thermostable lipase showed elevated activity when pretreated with BaCl2, CaCl2, and KCl with 112%, 108%, and 106%, respectively. Lipase hydrolyzed tripalmitin (C16 and olive oil with optimal activity (100% compared to other substrates.

Highly thermostable xylanase production from a thermophilic Geobacillus sp. strain WSUCF1 utilizing lignocellulosic biomass

Directory of Open Access Journals (Sweden)

Aditya eBhalla

2015-06-01

Full Text Available AbstractEfficient enzymatic hydrolysis of lignocellulose to fermentable sugars requires a complete repertoire of biomass deconstruction enzymes. Hemicellulases play an important role in hydrolyzing hemicellulose component of lignocellulose to xylo-oligosaccharides and xylose. Thermostable xylanases have been a focus of attention as industrially important enzymes due to their long shelf life at high temperatures. Geobacillus sp. strain WSUCF1 produced thermostable xylanase activity (crude xylanase cocktail when grown on xylan or various inexpensive untreated and pretreated lignocellulosic biomasses such as prairie cord grass and corn stover. The optimum pH and temperature for the crude xylanase cocktail were 6.5 and 70ºC, respectively. The WSUCF1 crude xylanase was found to be highly thermostable with half-lives of 18 and 12 days at 60 and 70ºC, respectively. At 70ºC, rates of xylan hydrolysis were also found to be better with the WSUCF1 secretome than those with commercial enzymes, i.e., for WSUCF1 crude xylanase, CellicHTec2, and AccelleraseXY, the percent xylan conversions were 68.9, 49.4, and 28.92, respectively. To the best of our knowledge, WSUCF1 crude xylanase cocktail is among the most thermostable xylanases produced by thermophilic Geobacillus spp. and other thermophilic microbes (optimum growth temperature ≤70ºC. High thermostability, activity over wide range of temperatures, and better xylan hydrolysis than commercial enzymes make WSUCF1 crude xylanase suitable for thermophilic lignocellulose bioconversion processes.

A virosomal formulated Her-2/neu multi-peptide vaccine induces Her-2/neu-specific immune responses in patients with metastatic breast cancer: a phase I study.

Science.gov (United States)

Wiedermann, Ursula; Wiltschke, C; Jasinska, J; Kundi, M; Zurbriggen, R; Garner-Spitzer, E; Bartsch, R; Steger, G; Pehamberger, H; Scheiner, O; Zielinski, C C

2010-02-01

We have previously shown in mice that vaccination with three Her-2-peptides representing B-cell epitopes of the extracellular domain of Her-2/neu induces Her-2/neu-specific IgG antibodies with strong anti-tumor activity in vitro and in vivo. We have now finalized a phase I clinical trial with an anti-Her-2/neu vaccine-construct of immunopotentiating reconstituted influenza virosomes with the three peptides in patients with metastatic breast cancer (MBC). Ten MBC patients with low protein overexpression of Her-2/neu of MBC (+ or ++ upon immunohistochemistry, FISH negative) and positive hormone receptor status were enrolled in a single center phase I study. The virosomal formulated vaccine, consisting of 10 microg/peptide, was intramuscularly applied three times on days 1, 28, and 56. The primary endpoint of the study, which lasted 12 weeks, was safety, the secondary endpoint immunogenicity. Local erythema at the injection site was the only vaccine-related side effect occurring in four patients. In 8 of 10 patients an increase in peptide-specific antibody titer measured by ELISA was found. Importantly, the induced antibodies were also directed against the native Her-2/neu protein. Cellular immune responses, as measured by in vitro production of IL-2, IFN-c, and TNF-a of PBMCs showed a marked increase after vaccination in the majority of vaccinees. Notably, the number of CD4+CD25+Foxp3+T regulatory cells, which were significantly increased compared to healthy controls prior to vaccination, was markedly reduced following vaccination. In all, the immunological responses after vaccination indicated that the patients in stage IV of disease were immunocompetent and susceptible to vaccination. The Her-2/neu multipeptide vaccine was safe, well tolerated and effective in overcoming immunological tolerance to Her-2/neu. The induction of anti-Her-2-specific antibodies could result in clinical benefit comparable to passive anti-Her-2 antibody therapy.

Construction and expression of immunogenic hybrid enterotoxigenic Escherichia coli CFA/I and CS2 colonization fimbriae for use in vaccines.

Science.gov (United States)

Tobias, Joshua; Svennerholm, Ann-Mari; Holmgren, Jan; Lebens, Michael

2010-07-01

Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrheal morbidity in developing countries, especially in children and also of traveler's diarrhea. Colonization factors (CFs) of ETEC, like CFA/I and CS2 which are genetically and structurally related, play a substantial role in pathogenicity, and since intestinal-mucosal immune responses against CFs appear to be protective, much effort has focused on the development of a CF-based ETEC vaccine. We have constructed hybrid operons in which the major CS2 subunit-encoding cotA gene was inserted into the CFA/I operon, either replacing (hybrid I) or being added to the major CFA/I subunit-encoding cfaB gene (hybrid II). Using specific monoclonal antibodies against the major subunits of CFA/I and CS2, high levels of surface expression of both fimbrial subunits were shown in E. coli carrying the hybrid II operon. Oral immunization of mice with formalin-killed bacteria expressing hybrid II fimbriae induced strong CFA/I- and CS2-specific serum IgG + IgM and fecal IgA antibody responses, which were higher than those achieved by similar immunization with the reference strains. Bacteria expressing hybrid fimbriae are potential candidate strains in an oral-killed CF-ETEC vaccine, and the approach represents an attractive and novel means of producing a broad-spectrum ETEC vaccine.

Carbohydrate-Based Ice Recrystallization Inhibitors Increase Infectivity and Thermostability of Viral Vectors

Science.gov (United States)

Ghobadloo, Shahrokh M.; Balcerzak, Anna K.; Gargaun, Ana; Muharemagic, Darija; Mironov, Gleb G.; Capicciotti, Chantelle J.; Briard, Jennie G.; Ben, Robert N.; Berezovski, Maxim V.

2014-07-01

The inability of vaccines to retain sufficient thermostability has been an obstacle to global vaccination programs. To address this major limitation, we utilized carbohydrate-based ice recrystallization inhibitors (IRIs) to eliminate the cold chain and stabilize the potency of Vaccinia virus (VV), Vesicular Stomatitis virus (VSV) and Herpes virus-1 (HSV-1). The impact of these IRIs was tested on the potency of the viral vectors using a plaque forming unit assay following room temperature storage, cryopreservation with successive freeze-thaw cycles and lyophilization. Viral potency after storage with all three conditions demonstrated that N-octyl-gluconamide (NOGlc) recovered the infectivity of shelf stored VV, 5.6 Log10 PFU mL-1 during 40 days, and HSV-1, 2.7 Log10 PFU mL-1 during 9 days. Carbon-linked antifreeze glycoprotein analogue ornithine-glycine-glycine-galactose (OGG-Gal) increases the recovery of VV and VSV more than 1 Log10 PFU mL-1 after 10 freeze-thaw cycles. In VSV, cryostorage with OGG-Gal maintains high infectivity and reduces temperature-induced aggregation of viral particles by 2 times that of the control. In total, OGG-Gal and NOGlc preserve virus potency during cryostorage. Remarkably, NOGlc has potential to eliminate the cold chain and permit room temperature storage of viral vectors.

Reversion to virulence and efficacy of an attenuated canarypox vaccine in Hawai'i 'Amakihi (Hemignathus Virens)

Science.gov (United States)

Atkinson, Carter T.; Wiegand, Kimberly C.; Triglia, Dennis; Jarvi, Susan I.

2012-01-01

Vaccines may be effective tools for protecting small populations of highly susceptible endangered, captive-reared, or translocated Hawaiian honeycreepers from introduced Avipoxvirus, but their efficacy has not been evaluated. An attenuated Canarypox vaccine that is genetically similar to one of two passerine Avipoxvirus isolates from Hawai‘i and distinct from Fowlpox was tested to evaluate whether Hawai‘i ‘Amakihi (Hemignathus virens) can be protected from wild isolates of Avipoxvirus from the Hawaiian Islands. Thirty-one (31) Hawai‘i ‘Amakihi were collected from high-elevation habitats on Mauna Kea Volcano, where pox transmission is rare, and randomly divided into two groups. One group was vaccinated with Poximune C®, whereas the other group received a sham vaccination with sterile water. Four of 15 (27%) vaccinated birds developed life-threatening disseminated lesions or lesions of unusually long duration, whereas one bird never developed a vaccine-associated lesion or “take.” After vaccine lesions healed, vaccinated birds were randomly divided into three groups of five and challenged with either a wild isolate of Fowlpox (FP) from Hawai‘i, a Hawai‘i ‘Amakihi isolate of a Canarypox-like virus (PV1), or a Hawai‘i ‘Amakihi isolate of a related, but distinct, passerine Avipoxvirus (PV2). Similarly, three random groups of five unvaccinated ‘Amakihi were challenged with the same virus isolates. Vaccinated and unvaccinated ‘Amakihi challenged with FP had transient infections with no clinical signs of infection. Mortality in vaccinated ‘Amakihi challenged with PV1 and PV2 ranged from 0% (0/5) for PV1 to 60% (3/5) for PV2. Mortality in unvaccinated ‘Amakihi ranged from 40% (2/5) for PV1 to 100% (5/5) for PV2. Although the vaccine provided some protection against PV1, both potential for vaccine reversion and low efficacy against PV2 preclude its use in captive or wild honeycreepers.

Tolerogenic β2-glycoprotein I DNA vaccine and FK506 as an adjuvant attenuates experimental obstetric antiphospholipid syndrome.

Science.gov (United States)

Chao, Ya-Hsuan; Chen, Der-Yuan; Lan, Joung-Liang; Tang, Kuo-Tung; Lin, Chi-Chien

2018-01-01

DNA vaccines have recently emerged as a therapeutic agent for treating autoimmune diseases, such as multiple sclerosis. Antiphospholipid antibody syndrome (APS) is an autoimmune disease characterized by β2-glycoprotein I (β2-GPI)-targeting antiphospholipid antibodies (APAs) and vascular thrombosis or obstetrical complications. To examine the therapeutic potential of a β2-GPI DNA vaccine, we administered a vaccine mixed with FK506 as an adjuvant to a mouse model of obstetric APS. First, the pCMV3-β2-GPI DNA vaccine, which encodes the full-length human β2-GPI gene, was constructed. Then, we administered the β2-GPI DNA vaccine in 0.1 ml of saline, mixed with or without 100 μg of FK506, intramuscularly to the mice on days 28, 35 and 42. Blood titers of the anti-β2-GPI antibody, platelet counts, activated partial thromboplastin times (aPTTs), and the percentage of fetal loss were measured. We also stimulated murine splenic T cells ex vivo with β2-GPI and determined the T helper cell proportion and cytokine secretion. The administration of the β2-GPI DNA vaccine mixed with FK506 reduced the blood IgG anti-β2-GPI antibody titers and suppressed APS manifestations in mice. The combination also suppressed interferon-γ and interleukin (IL)-17A secretion but increased the Treg cell proportion and IL-10 secretion in murine splenic T cells following ex vivo stimulation with β2-GPI. Our results demonstrated the therapeutic efficacy of a β2-GPI DNA vaccine and FK506 as an adjuvant in a murine model of obstetric APS. Possible mechanisms include the inhibition of Th1 and Th17 responses and the up-regulation of Treg cells.

Crystallization and preliminary X-ray crystallographic analysis of highly thermostable L2 lipase from the newly isolated Bacillus sp. L2

International Nuclear Information System (INIS)

Shariff, Fairolniza Mohd; Rahman, Raja Noor Zaliha Raja Abd.; Ali, Mohd Shukuri Mohamad; Chor, Adam Leow Thean; Basri, Mahiran; Salleh, Abu Bakar

2010-01-01

Thermostable recombinant L2 lipase from thermophilic Bacillus sp. L2 has been crystallized by using counter-diffusion method and diffracted to 2.7 Å resolution. The crystal belongs to the primitive orthorhombic space group P2 1 2 1 2 1 with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 Å. Purified thermostable recombinant L2 lipase from Bacillus sp. L2 was crystallized by the counter-diffusion method using 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl as precipitant. X-ray diffraction data were collected to 2.7 Å resolution using an in-house Bruker X8 PROTEUM single-crystal diffractometer system. The crystal belonged to the primitive orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 Å. The asymmetric unit contained one single molecule of protein, with a Matthews coefficient (V M ) of 2.85 Å 3 Da −1 and a solvent content of 57%

Oral vaccination of wildlife using a vaccinia-rabies-glycoprotein recombinant virus vaccine (RABORAL V-RG®): a global review.

Science.gov (United States)

Maki, Joanne; Guiot, Anne-Laure; Aubert, Michel; Brochier, Bernard; Cliquet, Florence; Hanlon, Cathleen A; King, Roni; Oertli, Ernest H; Rupprecht, Charles E; Schumacher, Caroline; Slate, Dennis; Yakobson, Boris; Wohlers, Anne; Lankau, Emily W

2017-09-22

RABORAL V-RG ® is an oral rabies vaccine bait that contains an attenuated ("modified-live") recombinant vaccinia virus vector vaccine expressing the rabies virus glycoprotein gene (V-RG). Approximately 250 million doses have been distributed globally since 1987 without any reports of adverse reactions in wildlife or domestic animals since the first licensed recombinant oral rabies vaccine (ORV) was released into the environment to immunize wildlife populations against rabies. V-RG is genetically stable, is not detected in the oral cavity beyond 48 h after ingestion, is not shed by vaccinates into the environment, and has been tested for thermostability under a range of laboratory and field conditions. Safety of V-RG has been evaluated in over 50 vertebrate species, including non-human primates, with no adverse effects observed regardless of route or dose. Immunogenicity and efficacy have been demonstrated under laboratory and field conditions in multiple target species (including fox, raccoon, coyote, skunk, raccoon dog, and jackal). The liquid vaccine is packaged inside edible baits (i.e., RABORAL V-RG, the vaccine-bait product) which are distributed into wildlife habitats for consumption by target species. Field application of RABORAL V-RG has contributed to the elimination of wildlife rabies from three European countries (Belgium, France and Luxembourg) and of the dog/coyote rabies virus variant from the United States of America (USA). An oral rabies vaccination program in west-central Texas has essentially eliminated the gray fox rabies virus variant from Texas with the last case reported in a cow during 2009. A long-term ORV barrier program in the USA using RABORAL V-RG is preventing substantial geographic expansion of the raccoon rabies virus variant. RABORAL V-RG has also been used to control wildlife rabies in Israel for more than a decade. This paper: (1) reviews the development and historical use of RABORAL V-RG; (2) highlights wildlife rabies control

Phase I trial of RV3-BB rotavirus vaccine: a human neonatal rotavirus vaccine.

Science.gov (United States)

Danchin, M; Kirkwood, C D; Lee, K J; Bishop, R F; Watts, E; Justice, F A; Clifford, V; Cowley, D; Buttery, J P; Bines, J E

2013-05-28

RV3 is a human neonatal rotavirus strain (G3P[6]) that has been associated with asymptomatic neonatal infection and replicates well in the infant gut. RV3-BB rotavirus vaccine has been developed as a rotavirus vaccine candidate for administration at birth. A single-centre, double-blind, randomised placebo-controlled Phase I study evaluated the safety and tolerability of a single oral dose of the second generation RV3-BB rotavirus vaccine (8.3×10(6)FFU/mL) in 20 adults, 20 children and 20 infants (10 vaccine and 10 placebo per age cohort). Vaccine take was defined as seroconversion (a 3-fold increase in serum anti-rotavirus IgA or serum neutralising antibody (SNA) from baseline at day 28 post-dose) or evidence of RV3-BB viral replication in the faeces by RT-PCR analysis 3-6 days post-vaccination. RV3-BB presence was confirmed by sequence analysis. The RV3-BB vaccine was well tolerated in all participants, with no pattern of adverse events shown to be associated with the study vaccine. In the infant cohort, vaccine take was demonstrated in 8/9 infants following a single dose of vaccine compared with 2/7 placebo recipients. In the infant vaccine group, 5/9 infants exhibited either IgA or SNA seroconversion and 7/9 infants had evidence of RV3-BB replication on days 3-6, compared with 2/7 infants who seroconverted and 0/10 infants with evidence of replication in the placebo group. Two infants in the placebo group had serological evidence of a rotavirus infection within the 28-day study period: one demonstrated an IgA and the other an SNA response, with wild-type virus replication detected in another infant. A single dose of RV3-BB rotavirus vaccine was well tolerated in adults, children and infants. Most infants (8/9) who received RV3-BB demonstrated vaccine take following a single dose. These data support progression of RV3-BB to Phase II immunogenicity and efficacy trials. Copyright © 2013 Elsevier Ltd. All rights reserved.

Subtype C gp140 Vaccine Boosts Immune Responses Primed by the South African AIDS Vaccine Initiative DNA-C2 and MVA-C HIV Vaccines after More than a 2-Year Gap.

Science.gov (United States)

Gray, Glenda E; Mayer, Kenneth H; Elizaga, Marnie L; Bekker, Linda-Gail; Allen, Mary; Morris, Lynn; Montefiori, David; De Rosa, Stephen C; Sato, Alicia; Gu, Niya; Tomaras, Georgia D; Tucker, Timothy; Barnett, Susan W; Mkhize, Nonhlanhla N; Shen, Xiaoying; Downing, Katrina; Williamson, Carolyn; Pensiero, Michael; Corey, Lawrence; Williamson, Anna-Lise

2016-06-01

A phase I safety and immunogenicity study investigated South African AIDS Vaccine Initiative (SAAVI) HIV-1 subtype C (HIV-1C) DNA vaccine encoding Gag-RT-Tat-Nef and gp150, boosted with modified vaccinia Ankara (MVA) expressing matched antigens. Following the finding of partial protective efficacy in the RV144 HIV vaccine efficacy trial, a protein boost with HIV-1 subtype C V2-deleted gp140 with MF59 was added to the regimen. A total of 48 participants (12 U.S. participants and 36 Republic of South Africa [RSA] participants) were randomized to receive 3 intramuscular (i.m.) doses of SAAVI DNA-C2 of 4 mg (months 0, 1, and 2) and 2 i.m. doses of SAAVI MVA-C of 1.45 × 10(9) PFU (months 4 and 5) (n = 40) or of a placebo (n = 8). Approximately 2 years after vaccination, 27 participants were rerandomized to receive gp140/MF59 at 100 μg or placebo, as 2 i.m. injections, 3 months apart. The vaccine regimen was safe and well tolerated. After the DNA-MVA regimen, CD4(+) T-cell and CD8(+) T-cell responses occurred in 74% and 32% of the participants, respectively. The protein boost increased CD4(+) T-cell responses to 87% of the subjects. All participants developed tier 1 HIV-1C neutralizing antibody responses as well as durable Env binding antibodies that recognized linear V3 and C5 peptides. The HIV-1 subtype C DNA-MVA vaccine regimen showed promising cellular immunogenicity. Boosting with gp140/MF59 enhanced levels of binding and neutralizing antibodies as well as CD4(+) T-cell responses to HIV-1 envelope. (This study has been registered at ClinicalTrials.gov under registration no. NCT00574600 and NCT01423825.). Copyright © 2016 Gray et al.

Vaccines to Combat Livestock Diseases in Sub-Saharan Africa ...

International Development Research Centre (IDRC) Digital Library (Canada)

This project is applying modern biotechnology to engineer a thermostable, single dose vaccine that protects cattle, sheep, and goats from five main diseases - lumpy skin ... In partnership with UNESCO's Organization for Women in Science for the Developing World (OWSD), IDRC is pleased to announce that the first call for ...

Thermostable enzymes as biocatalysts in the biofuel industry.

Science.gov (United States)

Yeoman, Carl J; Han, Yejun; Dodd, Dylan; Schroeder, Charles M; Mackie, Roderick I; Cann, Isaac K O

2010-01-01

Lignocellulose is the most abundant carbohydrate source in nature and represents an ideal renewable energy source. Thermostable enzymes that hydrolyze lignocellulose to its component sugars have significant advantages for improving the conversion rate of biomass over their mesophilic counterparts. We review here the recent literature on the development and use of thermostable enzymes for the depolymerization of lignocellulosic feedstocks for biofuel production. Furthermore, we discuss the protein structure, mechanisms of thermostability, and specific strategies that can be used to improve the thermal stability of lignocellulosic biocatalysts. Copyright 2010 Elsevier Inc. All rights reserved.

Enhancing activity and thermostability of lipase A from Serratia marcescens by site-directed mutagenesis.

Science.gov (United States)

Mohammadi, Mohsen; Sepehrizadeh, Zargham; Ebrahim-Habibi, Azadeh; Shahverdi, Ahmad Reza; Faramarzi, Mohammad Ali; Setayesh, Neda

2016-11-01

Lipases as significant biocatalysts had been widely employed to catalyze various chemical reactions such as ester hydrolysis, ester synthesis, and transesterification. Improving the activity and thermostability of enzymes is desirable for industrial applications. The lipase of Serratia marcescens belonging to family I.3 lipase has a very important pharmaceutical application in production of chiral precursors. In the present study, to achieve improved lipase activity and thermostability, using computational predictions of protein, four mutant lipases of SML (MutG2P, MutG59P, Mut H279K and MutL613WA614P) were constructed by site-directed mutagenesis. The recombinant mutant proteins were over-expressed in E. coli and purified by affinity chromatography on the Ni-NTA system. Circular dichroism spectroscopy, differential scanning calorimetry and kinetic parameters (Km and kcat) were determined. Our results have shown that the secondary structure of all lipases was approximately similar to one another. The MutG2P and MutG59P were more stable than wild type by approximately 2.3 and 2.9 in T 1/2 , respectively. The catalytic efficiency (kcat/Km) of MutH279K was enhanced by 2-fold as compared with the wild type (p<0.05). These results indicate that using protein modeling program and creating mutation, can enhance lipase activity and/or thermostability of SML and it also could be used for improving other properties of enzyme to the desired requirements as well as further mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

Hepatitis B surface antigen incorporated in dissolvable microneedle array patch is antigenic and thermostable.

Science.gov (United States)

Poirier, Danielle; Renaud, Frédéric; Dewar, Vincent; Strodiot, Laurent; Wauters, Florence; Janimak, Jim; Shimada, Toshio; Nomura, Tatsuya; Kabata, Koki; Kuruma, Koji; Kusano, Takayuki; Sakai, Masaki; Nagasaki, Hideo; Oyamada, Takayoshi

2017-11-01

Alternatives to syringe-based administration are considered for vaccines. Intradermal vaccination with dissolvable microneedle arrays (MNA) appears promising in this respect, as an easy-to-use and painless method. In this work, we have developed an MNA patch (MNAP) made of hydroxyethyl starch (HES) and chondroitin sulphate (CS). In swines, hepatitis B surface antigen (HBsAg) formulated with the saponin QS-21 as adjuvant, both incorporated in HES-based MNAP, demonstrated the same level of immunogenicity as a commercially available aluminum-adjuvanted HBsAg vaccine, after two immunizations 28 days apart. MNAP application was associated with transient skin reactions (erythema, lump, scab), particularly evident when the antigen was delivered with the adjuvant. The thermostability of the adjuvanted antigen when incorporated in the HES-based matrix was also assessed by storing MNAP at 37, 45 or 50 °C for up to 6 months. We could demonstrate that antigenicity was retained at 37 and 45 °C and only a 10% loss was observed after 6 months at 50 °C. Our results are supportive of MNAP as an attractive alternative to classical syringe-based vaccination. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

HPV vaccination prevalence, parental barriers and motivators to vaccinating children in Hawai'i.

Science.gov (United States)

Dela Cruz, May Rose Isnec; Braun, Kathryn L; Tsark, Jo Ann Umilani; Albright, Cheryl Lynn; Chen, John J

2018-05-10

To determine the prevalence and barriers to human papillomavirus (HPV) vaccine uptake among 11-18 year olds in the Hawai'i's four major ethnic groups-Native Hawaiians, Filipinos, Japanese, and Caucasians. A telephone survey assessed parents' knowledge of HPV and the HPV vaccine, status of their child's HPV vaccine uptake, variables operationalizing the Health Belief Model, and barriers and motivators to uptake. Across the groups, 799 parents completed the survey. About 35% of daughters and 19% of sons had received all three shots. Although ethnic differences in vaccine uptake were seen in bivariate analysis (with significantly lower uptake in Filipino youth), in multivariable logistic regression analysis, only Caucasian parents were significantly less likely to start their sons on the HPV vaccine series compared with Japanese parents (reference group). Having heard about the vaccine, believing in its effectiveness, and older age of the child were also associated with vaccine uptake. Motivators for HPV vaccination were physician's recommendation and wanting to protect one's child. The primary barrier to uptake was lack of knowledge about the vaccine. Findings reinforce the fact that a physician's recommendation and receipt of information about the vaccine are strong motivators for parents to vaccinate their children, regardless of ethnicity.

Vaccine mod halthed testes i besætning

DEFF Research Database (Denmark)

Lauritsen, Klara Tølbøll

2012-01-01

Ny vaccine mod ledbetændelse forårsaget af Mycoplasma hyosynoviae testes nu hos 200 svin i en problembesætning. Håbet er færre halte svin og en nedbringelse af antibiotikaforbruget.......Ny vaccine mod ledbetændelse forårsaget af Mycoplasma hyosynoviae testes nu hos 200 svin i en problembesætning. Håbet er færre halte svin og en nedbringelse af antibiotikaforbruget....

Functional analysis of thermostable proteins involved in carbohydrate metabolism

NARCIS (Netherlands)

Akerboom, A.P.

2007-01-01

Thermostable proteins can resist temperature stress whilst keeping their integrity and functionality. In many cases, thermostable proteins originate from hyperthermophilic microorganisms that thrive in extreme environments. These systems are generally located close to geothermal (volcanic) activity,

Thermo-stable carbon nanotube-TiO_2 nanocompsite as electron highways in dye-sensitized solar cell produced by bio-nano-process

International Nuclear Information System (INIS)

Inoue, Ippei; Yasueda, Hisashi; Yamauchi, Hirofumi; Okamoto, Naofumi; Toyoda, Kenichi; Horita, Masahiro; Ishikawa, Yasuaki; Uraoka, Yukiharu; Yamashita, Ichiro

2015-01-01

We produced a thermostable TiO_2-(anatase)-coated multi-walled-carbon-nanotube (MWNT) nanocomposite for use in dye-sensitized solar cells (DSSCs) using biological supuramolecules as catalysts. We synthesized two different sizes of iron oxide nanoparticles (NPs) and arrayed the NPs on a silicon substrate utilizing two kinds of genetically modified cage-shaped proteins with silicon-binding peptide aptamers on their outer surfaces. Chemical vapor deposition (CVD) with the vapor–liquid-solid phase (VLS) method was applied to the substrate, and thermostable MWNTs with a diameter of 6 ± 1 nm were produced. Using a genetically modified cage-shaped protein with carbon-nanomaterials binding and Ti-mineralizing peptides as a catalyst, we were able to mineralize a titanium compound around the surface of the MWNT. The products were sintered, and thin TiO_2-layer-coated MWNTs nanocomoposites were successfully produced. Addition of a 0.2 wt% TiO_2-coated MWNT nanocomposite to a DSSC photoelectrode improved current density by 11% and decreased electric resistance by 20% compared to MWNT-free reference DSSCs. These results indicate that a nanoscale TiO_2-layer-coated thermostable MWNT structure produced by our mutant proteins works as a superior electron transfer highway within TiO_2 photoelectrodes. (paper)

Indoleamine 2,3-dioxygenase vaccination

DEFF Research Database (Denmark)

Andersen, Mads Hald; Svane, Inge Marie

2015-01-01

Indoleamine 2,3-dioxygenase (IDO) is an immunoregulatory enzyme. Remarkably, we discovered IDO-specific T cells that can influence adaptive immune reactions in patients with cancer. Further, a recent phase I clinical trial demonstrated long-lasting disease stabilization without toxicity in patien...... with non-small-cell lung cancer (NSCLC) who were vaccinated with an IDO-derived HLA-A2-restricted epitope....

Engineering Thermostable Microbial Xylanases Toward its Industrial Applications.

Science.gov (United States)

Kumar, Vishal; Dangi, Arun Kumar; Shukla, Pratyoosh

2018-03-01

Xylanases are one of the important hydrolytic enzymes which hydrolyze the β-1, 4 xylosidic linkage of the backbone of the xylan polymeric chain which consists of xylose subunits. Xylanases are mainly found in plant cell walls and are produced by several kinds of microorganisms such as fungi, bacteria, yeast, and some protozoans. The fungi are considered as most potent xylanase producers than that of yeast and bacteria. There is a broad series of industrial applications for the thermostable xylanase as an industrial enzyme. Thermostable xylanases have been used in a number of industries such as paper and pulp industry, biofuel industry, food and feed industry, textile industry, etc. The present review explores xylanase-substrate interactions using gene-editing tools toward the comprehension in improvement in industrial stability of xylanases. The various protein-engineering and metabolic-engineering methods have also been explored to improve operational stability of xylanase. Thermostable xylanases have also been used for improvement in animal feed nutritional value. Furthermore, they have been used directly in bakery and breweries, including a major use in paper and pulp industry as a biobleaching agent. This present review envisages some of such applications of thermostable xylanases for their bioengineering.

Microneedle patches for vaccination in developing countries.

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Arya, Jaya; Prausnitz, Mark R

2016-10-28

Millions of people die of infectious diseases each year, mostly in developing countries, which could largely be prevented by the use of vaccines. While immunization rates have risen since the introduction of the Expanded Program on Immunization (EPI), there remain major challenges to more effective vaccination in developing countries. As a possible solution, microneedle patches containing an array of micron-sized needles on an adhesive backing have been developed to be used for vaccine delivery to the skin. These microneedle patches can be easily and painlessly applied by pressing against the skin and, in some designs, do not leave behind sharps waste. The patches are single-dose, do not require reconstitution, are easy to administer, have reduced size to simplify storage, transportation and waste disposal, and offer the possibility of improved vaccine immunogenicity, dose sparing and thermostability. This review summarizes vaccination challenges in developing countries and discusses advantages that microneedle patches offer for vaccination to address these challenges. We conclude that microneedle patches offer a powerful new technology that can enable more effective vaccination in developing countries. Copyright © 2015. Published by Elsevier B.V.

A phase 2 randomized controlled trial of a multicomponent meningococcal serogroup B vaccine (I).

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Prymula, Roman; Esposito, Susanna; Zuccotti, Gian Vincenzo; Xie, Fang; Toneatto, Daniela; Kohl, Igor; Dull, Peter M

2014-01-01

The novel meningococcal serogroup B vaccine (4CMenB, Bexsero(®)), recently approved in Europe and Australia, may soon be included in routine infant immunization schedules, subject to guidance from national or regional recommending bodies. In the development of 4CMenB and consistent with other newly introduced vaccines, clinical studies have shown concomitant administration with routine infant vaccines induces an incremental increase in some reactions, including fever. As this may hinder acceptability, we examined the impact of prophylactic paracetamol on the occurrence of fever and other solicited reactions, as well as the immune responses to study vaccines, in a prospectively designed study. 4CMenB was administered as a 4-dose series at 2, 3, 4, and 12 months of age concomitantly with routine infant vaccines: DTaP-HBV-IPV/Hib and PCV7, with or without prophylactic paracetamol; a third group received MenC vaccine. Immune responses to 4CMenB were not decreased by the use of paracetamol prophylaxis and there were no clinically relevant effects on immune responses to routine vaccines. Occurrence of fever was higher in infants co-administered with 4CMenB compared with those given MenC vaccine, but was significantly decreased by prophylactic paracetamol, as were other solicited reactions to vaccination, both local and systemic. Co-administration of 4CMenB had an acceptable tolerability profile, with no withdrawals due to vaccination-related adverse events. Inclusion of 4CMenB in routine infant immunization schedules will be a major advance in the control of meningococcal disease, and our study indicates that by using paracetamol prophylaxis, post-vaccination reactions are reduced without clinically relevant negative consequences on vaccine immunogenicity.

Prediction of Protein Thermostability by an Efficient Neural Network Approach

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Jalal Rezaeenour

2016-10-01

Full Text Available Introduction: Manipulation of protein stability is important for understanding the principles that govern protein thermostability, both in basic research and industrial applications. Various data mining techniques exist for prediction of thermostable proteins. Furthermore, ANN methods have attracted significant attention for prediction of thermostability, because they constitute an appropriate approach to mapping the non-linear input-output relationships and massive parallel computing. Method: An Extreme Learning Machine (ELM was applied to estimate thermal behavior of 1289 proteins. In the proposed algorithm, the parameters of ELM were optimized using a Genetic Algorithm (GA, which tuned a set of input variables, hidden layer biases, and input weights, to and enhance the prediction performance. The method was executed on a set of amino acids, yielding a total of 613 protein features. A number of feature selection algorithms were used to build subsets of the features. A total of 1289 protein samples and 613 protein features were calculated from UniProt database to understand features contributing to the enzymes’ thermostability and find out the main features that influence this valuable characteristic. Results:At the primary structure level, Gln, Glu and polar were the features that mostly contributed to protein thermostability. At the secondary structure level, Helix_S, Coil, and charged_Coil were the most important features affecting protein thermostability. These results suggest that the thermostability of proteins is mainly associated with primary structural features of the protein. According to the results, the influence of primary structure on the thermostabilty of a protein was more important than that of the secondary structure. It is shown that prediction accuracy of ELM (mean square error can improve dramatically using GA with error rates RMSE=0.004 and MAPE=0.1003. Conclusion: The proposed approach for forecasting problem

Immune-tolerant elastin-like polypeptides (iTEPs) and their application as CTL vaccine carriers.

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Cho, S; Dong, S; Parent, K N; Chen, M

2016-01-01

Cytotoxic T lymphocyte (CTL) vaccine carriers are known to enhance the efficacy of vaccines, but a search for more effective carriers is warranted. Elastin-like polypeptides (ELPs) have been examined for many medical applications but not as CTL vaccine carriers. We aimed to create immune tolerant ELPs using a new polypeptide engineering practice and create CTL vaccine carriers using the ELPs. Four sets of novel ELPs, termed immune-tolerant elastin-like polypeptide (iTEP) were generated according to the principles dictating humoral immunogenicity of polypeptides and phase transition property of ELPs. The iTEPs were non-immunogenic in mice. Their phase transition feature was confirmed through a turbidity assay. An iTEP nanoparticle (NP) was assembled from an amphiphilic iTEP copolymer plus a CTL peptide vaccine, SIINFEKL. The NP facilitated the presentation of the vaccine by dendritic cells (DCs) and enhanced vaccine-induced CTL responses. A new ELP design and development practice was established. The non-canonical motif and the immune tolerant nature of the iTEPs broaden our insights about ELPs. ELPs, for the first time, were successfully used as carriers for CTL vaccines. It is feasible to concurrently engineer both immune-tolerant and functional peptide materials. ELPs are a promising type of CTL vaccine carriers.

Thermostable cellulases, and mutants thereof, capable of hydrolyzing cellulose in ionic liquid

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Sapra, Rajat; Datta, Supratim; Chen, Zhiwei; Holmes, Bradley M.; Simmons, Blake A.; Blanch, Harvey W.

2016-04-26

The present invention provides for a composition comprising an ionic liquid and a thermostable cellulose, and a method of hydrolyzing a cellulose, comprising: (a) providing a composition comprising a solution comprising an ionic liquid and a cellulose, and (b) introducing a thermostable cellulase to the solution, such that the cellulose is hydrolyzed by the cellulase. The present invention also provides for a Thermatoga maritima thermostable cellulase mutant with increased cellulase activity.

Impact of PGL-I seropositivity on the protective effect of BCG vaccination among leprosy contacts: a cohort study.

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Nádia C Düppre

Full Text Available BACKGROUND: Contacts of leprosy patients are at increased risk of developing leprosy and need to be targeted for early diagnosis. Seropositivity to the phenolic glycolipid I (PGL-I antigen of Mycobacterium leprae has been used to identify contacts who have an increased risk of developing leprosy. In the present study, we studied the effect of seropositivity in patient contacts, on the risk of developing leprosy, stratified by Bacille Calmette Guerin (BCG vaccination after index case diagnosis. METHODOLOGY/PRINCIPAL FINDINGS: Leprosy contacts were examined as part of the surveillance programme of the Oswaldo Cruz Institute Leprosy Outpatient Clinic in Rio de Janeiro. Demographic, social, epidemiological and clinical data were collected. The presence of IgM antibodies to PGL-I in sera and BCG vaccination status at the time of index case diagnosis were evaluated in 2,135 contacts. During follow-up, 60 (2.8%; 60/2,135 leprosy cases were diagnosed: 41 among the 1,793 PGL-I-negative contacts and 19 among the 342 PGL-I-positive contacts. Among PGL-I-positive contacts, BCG vaccination after index case diagnosis increased the adjusted rate of developing clinical manifestations of leprosy (Adjusted Rate Ratio (aRR = 4.1; 95% CI: 1.8-8.2 compared with the PGL-I-positive unvaccinated contacts (aRR = 3.2; 95% CI: 1.2-8.1. The incidence density was highest during the first year of follow-up for the PGL-I-positive vaccinated contacts. However, all of those contacts developed PB leprosy, whereas most MB cases (4/6 occurred in PGL-I-positive unvaccinated contacts. CONCLUSION: Contact examination combined with PGL-I testing and BCG vaccination remain important strategies for leprosy control. The finding that rates of leprosy cases were highest among seropositive contacts justifies targeting this specific group for close monitoring. Furthermore, it is recommended that PGL-I-positive contacts and contacts with a high familial bacteriological index

Impact of PGL-I Seropositivity on the Protective Effect of BCG Vaccination among Leprosy Contacts: A Cohort Study

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Düppre, Nádia C.; Camacho, Luiz Antonio B.; Sales, Anna M.; Illarramendi, Ximena; Nery, José Augusto C.; Sampaio, Elizabeth P.; Sarno, Euzenir N.; Bührer-Sékula, Samira

2012-01-01

Background Contacts of leprosy patients are at increased risk of developing leprosy and need to be targeted for early diagnosis. Seropositivity to the phenolic glycolipid I (PGL-I) antigen of Mycobacterium leprae has been used to identify contacts who have an increased risk of developing leprosy. In the present study, we studied the effect of seropositivity in patient contacts, on the risk of developing leprosy, stratified by Bacille Calmette Guerin (BCG) vaccination after index case diagnosis. Methodology/Principal Findings Leprosy contacts were examined as part of the surveillance programme of the Oswaldo Cruz Institute Leprosy Outpatient Clinic in Rio de Janeiro. Demographic, social, epidemiological and clinical data were collected. The presence of IgM antibodies to PGL-I in sera and BCG vaccination status at the time of index case diagnosis were evaluated in 2,135 contacts. During follow-up, 60 (2.8%; 60/2,135) leprosy cases were diagnosed: 41 among the 1,793 PGL-I-negative contacts and 19 among the 342 PGL-I-positive contacts. Among PGL-I-positive contacts, BCG vaccination after index case diagnosis increased the adjusted rate of developing clinical manifestations of leprosy (Adjusted Rate Ratio (aRR) = 4.1; 95% CI: 1.8–8.2) compared with the PGL-I-positive unvaccinated contacts (aRR = 3.2; 95% CI: 1.2–8.1). The incidence density was highest during the first year of follow-up for the PGL-I-positive vaccinated contacts. However, all of those contacts developed PB leprosy, whereas most MB cases (4/6) occurred in PGL-I-positive unvaccinated contacts. Conclusion Contact examination combined with PGL-I testing and BCG vaccination remain important strategies for leprosy control. The finding that rates of leprosy cases were highest among seropositive contacts justifies targeting this specific group for close monitoring. Furthermore, it is recommended that PGL-I-positive contacts and contacts with a high familial bacteriological index, regardless of

Human Papillomavirus (HPV) Vaccination Motivators, Barriers, and Brochure Preferences Among Parents in Multicultural Hawai'i: a Qualitative Study.

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Dela Cruz, May Rose Isnec; Tsark, Jo Ann Umilani; Chen, John Jiangtian; Albright, Cheryl Lynn; Braun, Kathryn Lenzner

2017-09-01

The human papillomavirus (HPV) vaccine can prevent cervical and other cancers. Unfortunately, according to the National Immunization Survey-Teen 2014 data, completion of the HPV vaccine was only 38 % for 13- to 17-year-old girls and 31 % for 13- to17-year-old boys in the USA, and prevalence was similar in Hawai'i. Parents' acceptability of the HPV vaccine is critical for the vaccine uptake, and this can be increased by educational materials and interventions. However, HPV materials are not widely distributed in Hawai'i. The purpose of this qualitative study was to identify HPV vaccination barriers, motivators, and brochure preferences among parents of teens in multicultural Hawai'i. Twenty parents were interviewed in person or by telephone. Four major themes emerged: (1) the physician is critical in the decision to vaccinate, (2) parental perception of the child's sexual activity guides the timing of their willingness to vaccinate, (3) HPV health education materials should be provided and discussed by the physician, and (4) parents would prefer an educational brochure that features local faces and testimonials, includes an immunization chart, and addresses barriers to vaccination. These findings informed the development of HPV health education materials tailored to Asian Americans and Pacific Islanders in Hawai'i.

Application of the thermofluor PaSTRy technique for improving foot-and-mouth disease virus vaccine formulation.

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Kotecha, Abhay; Zhang, Fuquan; Juleff, Nicholas; Jackson, Terry; Perez, Eva; Stuart, Dave; Fry, Elizabeth; Charleston, Bryan; Seago, Julian

2016-07-01

Foot-and-mouth disease (FMD) has a major economic impact throughout the world and is a considerable threat to food security. Current FMD virus (FMDV) vaccines are made from chemically inactivated virus and need to contain intact viral capsids to maximize efficacy. FMDV exists as seven serotypes, each made up by a number of constantly evolving subtypes. A lack of immunological cross-reactivity between serotypes and between some strains within a serotype greatly complicates efforts to control FMD by vaccination. Thus, vaccines for one serotype do not afford protection against the others, and multiple-serotype-specific vaccines are required for effective control. The FMDV serotypes exhibit variation in their thermostability, and the capsids of inactivated preparations of the O, C and SAT serotypes are particularly susceptible to dissociation at elevated temperature. Methods to quantify capsid stability are currently limited, lack sensitivity and cannot accurately reflect differences in thermostability. Thus, new, more sensitive approaches to quantify capsid stability would be of great value for the production of more stable vaccines and to assess the effect of production conditions on vaccine preparations. Here we have investigated the application of a novel methodology (termed PaSTRy) that utilizes an RNA-binding fluorescent dye and a quantitative (q)PCR machine to monitor viral genome release and hence dissociation of the FMDV capsid during a slow incremental increase in temperature. PaSTRy was used to characterize capsid stability of all FMDV serotypes. Furthermore, we have used this approach to identify stabilizing factors for the most labile FMDV serotypes.

Thermostability of biological systems: fundamentals, challenges, and quantification.

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He, Xiaoming

2011-01-01

This review examines the fundamentals and challenges in engineering/understanding the thermostability of biological systems over a wide temperature range (from the cryogenic to hyperthermic regimen). Applications of the bio-thermostability engineering to either destroy unwanted or stabilize useful biologicals for the treatment of diseases in modern medicine are first introduced. Studies on the biological responses to cryogenic and hyperthermic temperatures for the various applications are reviewed to understand the mechanism of thermal (both cryo and hyperthermic) injury and its quantification at the molecular, cellular and tissue/organ levels. Methods for quantifying the thermophysical processes of the various applications are then summarized accounting for the effect of blood perfusion, metabolism, water transport across cell plasma membrane, and phase transition (both equilibrium and non-equilibrium such as ice formation and glass transition) of water. The review concludes with a summary of the status quo and future perspectives in engineering the thermostability of biological systems.

A Phase I Trial of a Human Papillomavirus (HPV) DNA Vaccine for HPV16+ Cervical Intraepithelial Neoplasia 2/3

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Trimble, Cornelia L.; Peng, Shiwen; Kos, Ferdynand; Gravitt, Patti; Viscidi, Raphael; Sugar, Elizabeth; Pardoll, Drew; Wu, TC

2010-01-01

Purpose: To evaluate the safety and immunogenicity of a therapeutic HPV16 DNA vaccine administered to women with HPV16+CIN2/3. Experimental Design: This phase I trial incorporated the standard ‘3+3” dose escalation design with an additional 6 patients allocated to the maximally tolerated dose (MTD). Healthy adult women with colposcopically-directed biopsy-proven HPV16+ CIN2/3 received three intramuscular (IM) vaccinations (0.5 mg, 1 mg, or 3mg) of a plasmid expressing a Sig-E7(detox)-HSP70 fusion protein on days 0, 28 and 56, and underwent standard therapeutic resection of the cervical squamocolumnar junction at day 105 (week 15). Safety and immunogenicity of the vaccine and histologic outcome based on resection at week 15 were assessed. Results: Fifteen patients were evaluable (3 each at 0.5 mg and 1mg, 9 at 3mg). The vaccine was well tolerated: most adverse events were mild transient injection-site discomfort; no dose-limiting toxicities were observed. Although HPVE7-specific T-cell responses to E7 detected by enzyme-linked immunospot assays (IFNγ) were of low frequency and magnitude, detectable increases in response subsequent to vaccination were identified in subjects in the second and third cohorts. Complete histologic regression occurred in 3/9 (33%, CI: 7%-70%)) individuals in the highest dose cohort, Although the difference is not significant, it is slightly higher than would be expected in an unvaccinated cohort (25%). Conclusions: This HPV16 DNA vaccine was safe and well tolerated. While it appears possible to elicit HPV-specific T cell responses in patients with established dysplastic lesions, other factors are likely to play a role in lesion regression. PMID:19118066

Molecular Dynamics Approach in Designing Thermostable Aspergillus niger Xylanase

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Malau, N. D.; Sianturi, M.

2017-03-01

Molecular dynamics methods we have applied as a tool in designing thermostable Aspergillus niger Xylanase, by examining Root Mean Square Deviation (RMSD) and The Stability of the Secondary Structure of enzymes structure at its optimum temperature and compare with its high temperature behavior. As RMSD represents structural fluctuation at a particular temperature, a better understanding of this factor will suggest approaches to bioengineer these enzymes to enhance their thermostability. In this work molecular dynamic simulations of Aspergillus niger xylanase (ANX) have been carried at 400K (optimum catalytic temperature) for 2.5 ns and 500K (ANX reported inactive temperature) for 2.5 ns. Analysis have shown that the Root Mean Square Deviation (RMSD) significant increase at higher temperatures compared at optimum temperature and some of the secondary structures of ANX that have been damaged at high temperature. Structural analysis revealed that the fluctuations of the α-helix and β-sheet regions are larger at higher temperatures compared to the fluctuations at optimum temperature.

Optimization of Thermostable Alpha-Amylase Production Via Mix Agricultural-Residues and <i>Bacillus amyloliquefaciensi>

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Shalini RAI

2014-03-01

Full Text Available This study reports utilization of mixture of wheat and barley bran (1:1 for the production of thermostable alpha-amylase enzyme through a spore former, heat tolerant strain of Bacillus amyloliquefaciens in solid state fermentation. Maximum yield of alpha-amylase (252.77 U mL-1 was obtained in following optimized conditions, inoculums size 2 mL (2 × 106 CFU/mL, moisture 80%, pH 7±0.02, NaCl (3%, temperature 38±1°C, incubation for 72 h, maltose (1% and tryptone (1%. After SSF crude enzyme was purified via ammonium sulfate precipitation, ion exchange and column chromatography by DEAE Cellulose. Purified protein showed a molecular weight of 42 kDa by SDS-PAGE electrophoresis. After purification, purified enzyme was characterized against several enzymes inhibitors such as temperature, NaCl, pH, metal and surfactants. Pure enzyme was highly active over broad temperature (50-70°C, NaCl concentration (0.5-4 M, and pH (6-10 ranges, indicating it’s a thermoactive and alkali-stable nature. Moreover, CaCl2, MnCl2, =-mercaptoethanol were found to stimulate the amylase activity, whereas FeCl3, sodium dodecyl sulfate (SDS, CuCl3 and ethylenediaminetetraacetic acid (EDTA strongly inhibited the enzyme. Moreover, enzyme specificity and thermal stability conformed by degradation of different soluble starch up to 55°C. Therefore, the present study proved that the extracellular alpha-amylase extracted through wheat flour residues by organism B. amyloliquefaciens MCCB0075, both have considerable potential for industrial application owing to its properties.

Draft genome sequence of a thermostable, alkaliphilic α-amylase and protease producing Bacillus amyloliquefaciens strain KCP2.

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Prajapati, Vimalkumar S; Ray, Sanket; Narayan, Jitendra; Joshi, Chaitanya C; Patel, Kamlesh C; Trivedi, Ujjval B; Patel, R M

2017-12-01

Bacillus amyloliquefaciens strain KCP2 was isolated from municipal food waste samples collected in Vallabh Vidyanagar, Gujarat, India. Strain KCP2 is noteworthy due to its ability to produce a thermostable, alkaliphilic α-amylase and a protease. These enzymes have importance in several industrial processes including bread making, brewing, starch processing, pharmacy, and textile industries. Whole genome sequencing of strain KCP2 showed that the estimated genome size was 3.9 Mb, the G + C content was 46%, and it coded for 4113 genes.

Low-dose priming before vaccination with the phase I chloroform-methanol residue vaccine against Q fever enhances humoral and cellular immune responses to Coxiella burnetii.

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Waag, David M; England, Marilyn J; Bolt, Christopher R; Williams, Jim C

2008-10-01

Although the phase I Coxiella burnetii cellular vaccine is completely efficacious in humans, adverse local and systemic reactions may develop if immune individuals are inadvertently vaccinated. The phase I chloroform-methanol residue (CMRI) vaccine was developed as a potentially safer alternative. Human volunteers with no evidence of previous exposure to C. burnetii received a subcutaneous vaccination with the CMRI vaccine in phase I studies under protocol IND 3516 to evaluate the safety and immunogenicity of the vaccine. This clinical trial tested escalating doses of the CMRI vaccine, ranging from 0.3 to 60 microg, followed by a booster dose of 30 microg, in a placebo-controlled study. Although priming doses of the CMRI vaccine did not induce a specific antibody detectable by enzyme-linked immunosorbent assay, booster vaccination stimulated the production of significant levels of anti-C. burnetii antibody. Peripheral blood cells (PBCs) of vaccinees responded to C. burnetii cellular antigen in vitro in a vaccine dose-dependent manner. After the booster dose, PBCs were activated by recall antigen in vitro, regardless of the priming dose. These findings suggest that vaccination with the CMRI vaccine can effectively prime the immune system to mount significant anamnestic responses after infection.

Immunodeficiency-related vaccine-derived poliovirus (iVDPV) cases: a systematic review and implications for polio eradication.

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Guo, Jean; Bolivar-Wagers, Sara; Srinivas, Nivedita; Holubar, Marisa; Maldonado, Yvonne

2015-03-03

Vaccine-derived polioviruses (VDPVs), strains of poliovirus mutated from the oral polio vaccine, pose a challenge to global polio eradication. Immunodeficiency-related vaccine-derived polioviruses (iVDPVs) are a type of VDPV which may serve as sources of poliovirus reintroduction after the eradication of wild-type poliovirus. This review is a comprehensive update of confirmed iVDPV cases published in the scientific literature from 1962 to 2012, and describes clinically relevant trends in reported iVDPV cases worldwide. We conducted a systematic review of published iVDPV case reports from January 1960 to November 2012 from four databases. We included cases in which the patient had a primary immunodeficiency, and the vaccine virus isolated from the patient either met the sequencing definition of VDPV (>1% divergence for serotypes 1 and 3 and >0.6% for serotype 2) and/or was previously reported as an iVDPV by the World Health Organization. We identified 68 iVDPV cases in 49 manuscripts reported from 25 countries and the Palestinian territories. 62% of case patients were male, 78% presented clinically with acute flaccid paralysis, and 65% were iVDPV2. 57% of cases occurred in patients with predominantly antibody immunodeficiencies, and the overall all-cause mortality rate was greater than 60%. The median age at case detection was 1.4 years [IQR: 0.8, 4.5] and the median duration of shedding was 1.3 years [IQR: 0.7, 2.2]. We identified a poliovirus genome VP1 region mutation rate of 0.72% per year and a higher median percent divergence for iVDPV1 cases. More cases were reported from high income countries, which also had a larger age variation and different distribution of immunodeficiencies compared to upper and lower middle-income countries. Our study describes the incidence and characteristics of global iVDPV cases reported in the literature in the past five decades. It also highlights the regional and economic disparities of reported iVDPV cases. Copyright © 2015

SAT2 Foot-and-Mouth Disease Virus Structurally Modified for Increased Thermostability.

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Scott, Katherine A; Kotecha, Abhay; Seago, Julian; Ren, Jingshan; Fry, Elizabeth E; Stuart, David I; Charleston, Bryan; Maree, Francois F

2017-05-15

Foot-and-mouth disease virus (FMDV), particularly strains of the O and SAT serotypes, is notoriously unstable. Consequently, vaccines derived from heat-labile SAT viruses have been linked to the induction of immunity with a poor duration and hence require more frequent vaccinations to ensure protection. In silico calculations predicted residue substitutions that would increase interactions at the interpentamer interface, supporting increased stability. We assessed the stability of the 18 recombinant mutant viruses in regard to their growth kinetics, antigenicity, plaque morphology, genetic stability, and temperature, ionic, and pH stability by using Thermofluor and inactivation assays in order to evaluate potential SAT2 vaccine candidates with improved stability. The most stable mutant for temperature and pH stability was the S2093Y single mutant, while other promising mutants were the E3198A, L2094V, and S2093H single mutants and the F2062Y-H2087M-H3143V triple mutant. Although the S2093Y mutant had the greatest stability, it exhibited smaller plaques, a reduced growth rate, a change in monoclonal antibody footprint, and poor genetic stability properties compared to those of the wild-type virus. However, these factors affecting production can be overcome. The addition of 1 M NaCl was found to further increase the stability of the SAT2 panel of viruses. The S2093Y and S2093H mutants were selected for future use in stabilizing SAT2 vaccines. IMPORTANCE Foot-and-mouth disease virus (FMDV) causes a highly contagious acute vesicular disease in cloven-hoofed livestock and wildlife. The control of the disease by vaccination is essential, especially at livestock-wildlife interfaces. The instability of some serotypes, such as SAT2, affects the quality of vaccines and therefore the duration of immunity. We have shown that we can improve the stability of SAT2 viruses by mutating residues at the capsid interface through predictive modeling. This is an important finding for

Structural characterization of the thermostable Bradyrhizobium japonicumD-sorbitol dehydrogenase.

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Fredslund, Folmer; Otten, Harm; Gemperlein, Sabrina; Poulsen, Jens Christian N; Carius, Yvonne; Kohring, Gert Wieland; Lo Leggio, Leila

2016-11-01

Bradyrhizobium japonicum sorbitol dehydrogenase is NADH-dependent and is active at elevated temperatures. The best substrate is D-glucitol (a synonym for D-sorbitol), although L-glucitol is also accepted, giving it particular potential in industrial applications. Crystallization led to a hexagonal crystal form, with crystals diffracting to 2.9 Å resolution. In attempts to phase the data, a molecular-replacement solution based upon PDB entry 4nbu (33% identical in sequence to the target) was found. The solution contained one molecule in the asymmetric unit, but a tetramer similar to that found in other short-chain dehydrogenases, including the search model, could be reconstructed by applying crystallographic symmetry operations. The active site contains electron density consistent with D-glucitol and phosphate, but there was not clear evidence for the binding of NADH. In a search for the features that determine the thermostability of the enzyme, the T m for the orthologue from Rhodobacter sphaeroides, for which the structure was already known, was also determined, and this enzyme proved to be considerably less thermostable. A continuous β-sheet is formed between two monomers in the tetramer of the B. japonicum enzyme, a feature not generally shared by short-chain dehydrogenases, and which may contribute to thermostability, as may an increased Pro/Gly ratio.

I-MOVE multicentre case-control study 2010/11 to 2014/15: Is there within-season waning of influenza type/subtype vaccine effectiveness with increasing time since vaccination?

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Kissling, Esther; Nunes, Baltazar; Robertson, Chris; Valenciano, Marta; Reuss, Annicka; Larrauri, Amparo; Cohen, Jean Marie; Oroszi, Beatrix; Rizzo, Caterina; Machado, Ausenda; Pitigoi, Daniela; Domegan, Lisa; Paradowska-Stankiewicz, Iwona; Buchholz, Udo; Gherasim, Alin; Daviaud, Isabelle; Horváth, Judit Krisztina; Bella, Antonino; Lupulescu, Emilia; O Donnell, Joan; Korczyńska, Monika; Moren, Alain

2016-04-21

Since the 2008/9 influenza season, the I-MOVE multicentre case-control study measures influenza vaccine effectiveness (VE) against medically-attended influenza-like-illness (ILI) laboratory confirmed as influenza. In 2011/12, European studies reported a decline in VE against influenza A(H3N2) within the season. Using combined I-MOVE data from 2010/11 to 2014/15 we studied the effects of time since vaccination on influenza type/subtype-specific VE. We modelled influenza type/subtype-specific VE by time since vaccination using a restricted cubic spline, controlling for potential confounders (age, sex, time of onset, chronic conditions). Over 10,000 ILI cases were included in each analysis of influenza A(H3N2), A(H1N1)pdm09 and B; with 4,759, 3,152 and 3,617 influenza positive cases respectively. VE against influenza A(H3N2) reached 50.6% (95% CI: 30.0-65.1) 38 days after vaccination, declined to 0% (95% CI: -18.1-15.2) from 111 days onwards. At day 54 VE against influenza A(H1N1)pdm09 reached 55.3% (95% CI: 37.9-67.9) and remained between this value and 50.3% (95% CI: 34.8-62.1) until season end. VE against influenza B declined from 70.7% (95% CI: 51.3-82.4) 44 days after vaccination to 21.4% (95% CI: -57.4-60.8) at season end. To assess if vaccination campaign strategies need revising more evidence on VE by time since vaccination is urgently needed.

Informing vaccine decision-making: A strategic multi-attribute ranking tool for vaccines-SMART Vaccines 2.0.

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Knobler, Stacey; Bok, Karin; Gellin, Bruce

2017-01-20

SMART Vaccines 2.0 software is being developed to support decision-making among multiple stakeholders in the process of prioritizing investments to optimize the outcomes of vaccine development and deployment. Vaccines and associated vaccination programs are one of the most successful and effective public health interventions to prevent communicable diseases and vaccine researchers are continually working towards expanding targets for communicable and non-communicable diseases through preventive and therapeutic modes. A growing body of evidence on emerging vaccine technologies, trends in disease burden, costs associated with vaccine development and deployment, and benefits derived from disease prevention through vaccination and a range of other factors can inform decision-making and investment in new and improved vaccines and targeted utilization of already existing vaccines. Recognizing that an array of inputs influences these decisions, the strategic multi-attribute ranking method for vaccines (SMART Vaccines 2.0) is in development as a web-based tool-modified from a U.S. Institute of Medicine Committee effort (IOM, 2015)-to highlight data needs and create transparency to facilitate dialogue and information-sharing among decision-makers and to optimize the investment of resources leading to improved health outcomes. Current development efforts of the SMART Vaccines 2.0 framework seek to generate a weighted recommendation on vaccine development or vaccination priorities based on population, disease, economic, and vaccine-specific data in combination with individual preference and weights of user-selected attributes incorporating valuations of health, economics, demographics, public concern, scientific and business, programmatic, and political considerations. Further development of the design and utility of the tool is being carried out by the National Vaccine Program Office of the Department of Health and Human Services and the Fogarty International Center of the

Low-Dose Priming before Vaccination with the Phase I Chloroform-Methanol Residue Vaccine against Q Fever Enhances Humoral and Cellular Immune Responses to Coxiella burnetii▿

Science.gov (United States)

Waag, David M.; England, Marilyn J.; Bolt, Christopher R.; Williams, Jim C.

2008-01-01

Although the phase I Coxiella burnetii cellular vaccine is completely efficacious in humans, adverse local and systemic reactions may develop if immune individuals are inadvertently vaccinated. The phase I chloroform-methanol residue (CMRI) vaccine was developed as a potentially safer alternative. Human volunteers with no evidence of previous exposure to C. burnetii received a subcutaneous vaccination with the CMRI vaccine in phase I studies under protocol IND 3516 to evaluate the safety and immunogenicity of the vaccine. This clinical trial tested escalating doses of the CMRI vaccine, ranging from 0.3 to 60 μg, followed by a booster dose of 30 μg, in a placebo-controlled study. Although priming doses of the CMRI vaccine did not induce a specific antibody detectable by enzyme-linked immunosorbent assay, booster vaccination stimulated the production of significant levels of anti-C. burnetii antibody. Peripheral blood cells (PBCs) of vaccinees responded to C. burnetii cellular antigen in vitro in a vaccine dose-dependent manner. After the booster dose, PBCs were activated by recall antigen in vitro, regardless of the priming dose. These findings suggest that vaccination with the CMRI vaccine can effectively prime the immune system to mount significant anamnestic responses after infection. PMID:18701647

Brunenders: a partially attenuated historic poliovirus type I vaccine strain.

Science.gov (United States)

Sanders, Barbara P; Liu, Ying; Brandjes, Alies; van Hoek, Vladimir; de Los Rios Oakes, Isabel; Lewis, John; Wimmer, Eckard; Custers, Jerome H H V; Schuitemaker, Hanneke; Cello, Jeronimo; Edo-Matas, Diana

2015-09-01

Brunenders, a type I poliovirus (PV) strain, was developed in 1952 by J. F. Enders and colleagues through serial in vitro passaging of the parental Brunhilde strain, and was reported to display partial neuroattenuation in monkeys. This phenotype of attenuation encouraged two vaccine manufacturers to adopt Brunenders as the type I component for their inactivated poliovirus vaccines (IPVs) in the 1950s, although today no licensed IPV vaccine contains Brunenders. Here we confirmed, in a transgenic mouse model, the report of Enders on the reduced neurovirulence of Brunenders. Although dramatically neuroattenuated relative to WT PV strains, Brunenders remains more virulent than the attenuated oral vaccine strain, Sabin 1. Importantly, the neuroattenuation of Brunenders does not affect in vitro growth kinetics and in vitro antigenicity, which were similar to those of Mahoney, the conventional type I IPV vaccine strain. We showed, by full nucleotide sequencing, that Brunhilde and Brunenders differ at 31 nucleotides, eight of which lead to amino acid changes, all located in the capsid. Upon exchanging the Brunenders capsid sequence with that of the Mahoney capsid, WT neurovirulence was regained in vivo, suggesting a role for the capsid mutations in Brunenders attenuation. To date, as polio eradication draws closer, the switch to using attenuated strains for IPV is actively being pursued. Brunenders preceded this novel strategy as a partially attenuated IPV strain, accompanied by decades of successful use in the field. Providing data on the attenuation of Brunenders may be of value in the further construction of attenuated PV strains to support the grand pursuit of the global eradication of poliomyelitis.

Problems of increasing of thermostability of highly permeable Ni-Zn ferrites and relative materials for telecommunications

Energy Technology Data Exchange (ETDEWEB)

Gonchar, A. E-mail: letyuk@mail.ru; Andreev, V.; Letyuk, L.; Shishkanov, A.; Maiorov, V

2003-01-01

The work considers ways of increasing of thermostability of ferrites of the basic systems NiO-ZnO-Fe{sub 2}O{sub 3} and MgO-ZnO-Fe{sub 2}O{sub 3} and relative materials for telecommunication. Sufficient results in increasing of the thermostability were achieved by doping Cu ions and controlling rejection of Fe{sub 2}O{sub 3} content from equimolar composition. These results allow to increase the Curie temperature to 130-140 deg. C for Ni-Zn ferrites with initial permeability 2000.

Rainbow trout (<i>Oncorhynchus mykissi>) immune response towards a recombinant vaccine targeting the parasitic ciliate <i>Ichthyophthirius multifiliisi>

DEFF Research Database (Denmark)

Jørgensen, Louise von Gersdorff; Kania, Per Walter; Rasmussen, Karina Juhl

2017-01-01

produced in E. coli and one in insect cells) were combined and injected i.p., and subsequently, protection and antibody responses were analysed. Both the experimental and the benchmark vaccine induced partial but significant protection against I. multifiliis when compared to control fish. Specific antibody...

Purification and Characterization of Thermostable Cellulase from ...

African Journals Online (AJOL)

Available online at http://www.tjpr.org ... Methods: Molecular community structure of the newly selected thermophilic bacterial ... Keywords: Thermostable cellulase, Sugarcane bagasse, Purification, Characterization, Hot spring ... Currently, one.

Thermostability in rubredoxin and its relationship to mechanical rigidity

Science.gov (United States)

Rader, A. J.

2010-03-01

The source of increased stability in proteins from organisms that thrive in extreme thermal environments is not well understood. Previous experimental and theoretical studies have suggested many different features possibly responsible for such thermostability. Many of these thermostabilizing mechanisms can be accounted for in terms of structural rigidity. Thus a plausible hypothesis accounting for this remarkable stability in thermophilic enzymes states that these enzymes have enhanced conformational rigidity at temperatures below their native, functioning temperature. Experimental evidence exists to both support and contradict this supposition. We computationally investigate the relationship between thermostability and rigidity using rubredoxin as a case study. The mechanical rigidity is calculated using atomic models of homologous rubredoxin structures from the hyperthermophile Pyrococcus furiosus and mesophile Clostridium pasteurianum using the FIRST software. A global increase in structural rigidity (equivalently a decrease in flexibility) corresponds to an increase in thermostability. Locally, rigidity differences (between mesophilic and thermophilic structures) agree with differences in protection factors.

Thermostability in rubredoxin and its relationship to mechanical rigidity

International Nuclear Information System (INIS)

Rader, A J

2010-01-01

The source of increased stability in proteins from organisms that thrive in extreme thermal environments is not well understood. Previous experimental and theoretical studies have suggested many different features possibly responsible for such thermostability. Many of these thermostabilizing mechanisms can be accounted for in terms of structural rigidity. Thus a plausible hypothesis accounting for this remarkable stability in thermophilic enzymes states that these enzymes have enhanced conformational rigidity at temperatures below their native, functioning temperature. Experimental evidence exists to both support and contradict this supposition. We computationally investigate the relationship between thermostability and rigidity using rubredoxin as a case study. The mechanical rigidity is calculated using atomic models of homologous rubredoxin structures from the hyperthermophile Pyrococcus furiosus and mesophile Clostridium pasteurianum using the FIRST software. A global increase in structural rigidity (equivalently a decrease in flexibility) corresponds to an increase in thermostability. Locally, rigidity differences (between mesophilic and thermophilic structures) agree with differences in protection factors

Evaluations on power ramp data of PWR fuels by FROST and THERMOST codes

International Nuclear Information System (INIS)

Murai, K.; Ogawa, S.; Nuno, H.; Kondo, Y.

1987-01-01

An evaluation is presented of power ramp data of Mitsubishi's PWR fuel rods tested in R-2, Studsvik, which was analysed by FROST and THERMOST codes. The analyses give good predictions for measured diameter changes and on-power rod elongations. The work indicates that FROST is capable of analysing both radial and axial pellet-cladding mechanism interaction (PCMI) appropriately, and that predicted states of PCMI (i.e. stress and strain which cannot be measured directly) are considered to be reliable. The ramp data used in the present analyses were obtained in two joint programmes with five Japanese PWR utilities (KEPCO, KYEPCO, SEPCO, HEPCO, and JAPCO). (UK)

Thermostable endoglucanases in the liquefaction of hydrothermally pretreated wheat straw

Directory of Open Access Journals (Sweden)

Siika-aho Matti

2011-01-01

Full Text Available Abstract Background Thermostable enzymes have several benefits in lignocellulose processing. In particular, they potentially allow the use of increased substrate concentrations (because the substrate viscosity decreases as the temperature increases, resulting in improved product yields and reduced capital and processing costs. A short pre-hydrolysis step at an elevated temperature using thermostable enzymes aimed at rapid liquefaction of the feedstock is seen as an attractive way to overcome the technical problems (such as poor mixing and mass transfer properties connected with high initial solid loadings in the lignocellulose to ethanol process. Results The capability of novel thermostable enzymes to reduce the viscosity of high-solid biomass suspensions using a real-time viscometric measurement method was investigated. Heterologously expressed enzymes from various thermophilic organisms were compared for their ability to liquefy the lignocellulosic substrate, hydrothermally pretreated wheat straw. Once the best enzymes were identified, the optimal temperatures for these enzymes to decrease substrate viscosity were compared. The combined hydrolytic properties of the thermostable preparations were tested in hydrolysis experiments. The studied mixtures were primarily designed to have good liquefaction potential, and therefore contained an enhanced proportion of the key liquefying enzyme, EGII/Cel5A. Conclusions Endoglucanases were shown to have a superior ability to rapidly reduce the viscosity of the 15% (w/w; dry matter hydrothermally pretreated wheat straw. Based on temperature profiling studies, Thermoascus aurantiacus EGII/Cel5A was the most promising enzyme for biomass liquefaction. Even though they were not optimized for saccharification, many of the thermostable enzyme mixtures had superior hydrolytic properties compared with the commercial reference enzymes at 55°C.

Thermophilic growth and enzymatic thermostability are polyphyletic traits within Chaetomiaceae.

Science.gov (United States)

van den Brink, Joost; Facun, Kryss; de Vries, Michel; Stielow, J Benjamin

2015-12-01

Thermophilic fungi have the potential to produce industrial-relevant thermostable enzymes, in particular for the degradation of plant biomass. Sordariales is one of the few fungal orders containing several thermophilic taxa, of which many have been associated with the production of thermostable enzymes. The evolutionary affiliation of Sordariales fungi, especially between thermophiles and non-thermophilic relatives, is however poorly understood. Phylogenetic analysis within the current study was based on sequence data, derived from a traditional Sanger and highly multiplexed targeted next generation sequencing approach of 45 isolates. The inferred phylogeny and detailed growth analysis rendered the trait 'thermophily' as polyphyletic within Chaetomiaceae (Sordariales, Sordariomycetes), and characteristic to: Myceliophthora spp., Thielavia terrestris, Chaetomium thermophilum, and Mycothermus thermophilus. Compared to mesophiles, the isolates within thermophilic taxa produced enzyme mixtures with the highest thermostability of known cellulase activities. Temperature profiles of the enzyme activities correlated strongly with the optimal growth temperatures of the isolates but not with their phylogenetic relationships. This strong correlation between growth and enzyme characteristics indicated that detailed analysis of growth does give predictive information on enzyme physiology. The variation in growth and enzyme characteristics reveals these fungi as an excellent platform to better understand fungal thermophily and enzyme thermostability. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Engineering of pectinolytic enzymes for enhanced thermostability

DEFF Research Database (Denmark)

Larsen, Dorte Møller

Conversion of waste materials into valuable compounds is promising concerning transformation of byproduct streams such as sugar beet and potato pulp. In order to obtain those compounds with reduced energy consumption, carbohydrate active enzymes can be used as catalysts. Sugar beet and potato pulp...... consist of pectin that can be converted into beneficial polymeric and oligomeric carbohydrates requiring enzymes such as pectin lyases, rhamnogalacturonan I (RGI) lyases, polygalacturonases and galactanases. Enzymatic conversion of such pectinaceous biomasses at high temperatures is advantageous...... as it gives rise to lower substrate viscosity, easier mixing, higher substrate solubility and lowers the risk of contamination. The overall objective of this thesis was to discover enzymes for degradation of RGI structures in pectin and further engineer for enhanced thermostability. The hypotheses were...

Influence of high temperature and ethanol on thermostable lignocellulolytic enzymes

DEFF Research Database (Denmark)

Skovgaard, Pernille Anastasia; Jørgensen, Henning

2013-01-01

the influence of temperature and ethanol on enzyme activity and stability in the distillation step, where most enzymes are inactivated due to high temperatures. Two enzyme mixtures, a mesophilic and a thermostable mixture, were exposed to typical process conditions [temperatures from 55 to 65 °C and up to 5...... % ethanol (w/v)] followed by specific enzyme activity analyses and SDS-PAGE. The thermostable and mesophilic mixture remained active at up to 65 and 55 °C, respectively. When the enzyme mixtures reached their maximum temperature limit, ethanol had a remarkable influence on enzyme activity, e.g., the more...... ethanol, the faster the inactivation. The reason could be the hydrophobic interaction of ethanol on the tertiary structure of the enzyme protein. The thermostable mixture was more tolerant to temperature and ethanol and could therefore be a potential candidate for recycling after distillation....

A microneedle patch containing measles vaccine is immunogenic in non-human primates.

Science.gov (United States)

Edens, Chris; Collins, Marcus L; Goodson, James L; Rota, Paul A; Prausnitz, Mark R

2015-09-08

Very high vaccination coverage is required to eliminate measles, but achieving high coverage can be constrained by the logistical challenges associated with subcutaneous injection. To simplify the logistics of vaccine delivery, a patch containing micron-scale polymeric needles was formulated to encapsulate the standard dose of measles vaccine (1000 TCID₅₀) and the immunogenicity of the microneedle patch was compared with subcutaneous injection in rhesus macaques. The microneedle patch was administered without reconstitution with diluent, dissolved in skin within 10 min, and caused only mild, transient skin erythema. Both groups of rhesus macaques generated neutralizing antibody responses to measles that were consistent with protection and the neutralizing antibody titers were equivalent. In addition, the microneedle patches maintained an acceptable level of potency after storage at elevated temperature suggesting improved thermostability compared to standard lyophilized vaccine. In conclusion, a measles microneedle patch vaccine was immunogenic in non-human primates, and this approach offers a promising delivery method that could help increase vaccination coverage. Copyright © 2015 Elsevier Ltd. All rights reserved.

Site-saturation mutagenesis of Glomerella cingulata cutinase gene for enhanced enzyme thermostability

Science.gov (United States)

Hanapi, Wan Nurhidayah Wan; Iuan-Sheau, Chin; Mahadi, Nor Muhammad; Murad, Abdul Munir Abdul; Bakar, Farah Diba Abu

2015-09-01

Cutinase is an important biocatalyst for various industrial applications. This enzyme which has dual functionality comparable to esterases and lipases, is efficient in the hydrolysis of soluble esters and emulsified triacylglycerols. Naturally-occurring enzymes usually have disadvantages when applied in non-natural catalysis due to Glomerella cingulata cutinase enzyme thermostability. It is postulated that by increasing the rigidity at certain amino acid positions showing high mobility based on the three-dimensional structure of G. cingulata cutinase, the improvement in thermostability will be achieved. The amino acid N82 of G. cingulata cutinase was selected based on its high B-factor value determined via the B-FITTER program. Megaprimer PCR was employed to introduce mutations at the chosen site by randomization using NNK degenerate primers. About 300 transformants were selected for screening of positive cutinase variants. The N82_V14 cutinase variant was observed to be more thermostable at an almost 2-fold increase when exposed at 50°C for 1 hr as compared to the wild-type enzyme. This study may provide valuable information regarding thermal stability of cutinases denaturation at high temperatures.

Optimization of medium composition for thermostable protease ...

African Journals Online (AJOL)

SERVER

2008-04-17

Apr 17, 2008 ... Optimization of the fermentation medium for maximization of thermostable neutral protease production by Bacillus sp. ..... Each contour curve represented an infinite number of combinations of two ..... Production in sea-water of.

Evaluation of thermostable enzymes for bioethanol processing

DEFF Research Database (Denmark)

Skovgaard, Pernille Anastasia

of fermentable sugars (glucose) as cellulose is tightly linked to hemicellulose and lignin. Lignocellulose is disrupted during pretreatment, but to degrade cellulose to single sugars, lignocellulolytic enzymes such as cellulases and hemicellulases are needed. Lignocellulolytic enzymes are costly...... for the ioethanol production, but the expenses can be reduced by using thermostable enzymes, which are known for their increased stability and inhibitor olerance. However, the advantage of using thermostable enzymes has not been studied thoroughly and more knowledge is needed for development of bioethanol processes....... Enzymes are added to the bioethanol process after pretreatment. For an efficient sugar and ethanol yield, the solids content of biomass is normally increased, which results in highly viscous slurries that are difficult to mix. Therefore, the first enzymatic challenge is to ensure rapid reduction...

Bursitis efter vaccination i skulderregionen

DEFF Research Database (Denmark)

Bliddal, Henning; Torp-Pedersen, Søren; Falk-Riecke, Birgit

2017-01-01

Shoulder pain may develop after vaccination in the deltoid area due to inadvertent injection of the vaccine into the subdeltoid bursa, which may be located close to the skin. As far as we know, such vaccination reactions occur more frequently than officially registered, and doctors may not be awa...

Improved vaccine protection against retrovirus infection after co-administration of adenoviral vectors encoding viral antigens and type I interferon subtypes

Directory of Open Access Journals (Sweden)

Groitl Peter

2011-09-01

Full Text Available Abstract Background Type I interferons (IFNs exhibit direct antiviral effects, but also distinct immunomodulatory properties. In this study, we analyzed type I IFN subtypes for their effect on prophylactic adenovirus-based anti-retroviral vaccination of mice against Friend retrovirus (FV or HIV. Results Mice were vaccinated with adenoviral vectors encoding FV Env and Gag proteins alone or in combination with vectors encoding IFNα1, IFNα2, IFNα4, IFNα5, IFNα6, IFNα9 or IFNβ. Only the co-administration of adenoviral vectors encoding IFNα2, IFNα4, IFNα6 and IFNα9 resulted in strongly improved immune protection of vaccinated mice from subsequent FV challenge infection with high control over FV-induced splenomegaly and reduced viral loads. The level of protection correlated with augmented virus-specific CD4+ T cell responses and enhanced antibody titers. Similar results were obtained when mice were vaccinated against HIV with adenoviral vectors encoding HIV Env and Gag-Pol in combination with various type I IFN encoding vectors. Here mainly CD4+ T cell responses were enhanced by IFNα subtypes. Conclusions Our results indicate that certain IFNα subtypes have the potential to improve the protective effect of adenovirus-based vaccines against retroviruses. This correlated with augmented virus-specific CD4+ T cell and antibody responses. Thus, co-expression of select type I IFNs may be a valuable tool for the development of anti-retroviral vaccines.

Tailoring DNA vaccines: designing strategies against HER2 positive cancers

Directory of Open Access Journals (Sweden)

Cristina eMarchini

2013-05-01

Full Text Available The crucial role of HER2 in epithelial transformation and its selective overexpression on cancer tissues makes it an ideal target for cancer immunotherapies such as passive immunotherapy with Trastuzumab. There are, however, a number of concerns regarding the use of monoclonal antibodies which include resistance, repeated treatments, considerable costs and side effects that make active immunotherapies against HER2 desirable alternative approaches. The efficacy of anti-HER2 DNA vaccination has been widely demonstrated in transgenic cancer-prone mice, which recapitulate several features of human breast cancers. Nonetheless, the rational design of a cancer vaccine able to trigger a long lasting immunity, and thus prevent tumor recurrence in patients, would require the understanding of how tolerance and immunosuppression regulate antitumor immune responses and, at the same time, the identification of the most immunogenic portions of the target protein. We herein retrace the findings that led to our most promising DNA vaccines that, by encoding human/rat chimeric forms of HER2, are able to circumvent peripheral tolerance. Preclinical data obtained with these chimeric DNA vaccines have provided the rationale for their use in an ongoing phase I clinical trial (EudraCT 2011-001104-34.

Directed Evolution of Recombinant C-Terminal Truncated Staphylococcus epidermidis Lipase AT2 for the Enhancement of Thermostability

Directory of Open Access Journals (Sweden)

Jiivittha Veno

2017-11-01

Full Text Available In the industrial processes, lipases are expected to operate at temperatures above 45 °C and could retain activity in organic solvents. Hence, a C-terminal truncated lipase from Staphylococcus epidermis AT2 (rT-M386 was engineered by directed evolution. A mutant with glycine-to-cysteine substitution (G210C demonstrated a remarkable improvement of thermostability, whereby the mutation enhanced the activity five-fold when compared to the rT-M386 at 50 °C. The rT-M386 and G210C lipases were purified concurrently using GST-affinity chromatography. The biochemical and biophysical properties of both enzymes were investigated. The G210C lipase showed a higher optimum temperature (45 °C and displayed a more prolonged half-life in the range of 40–60 °C as compared to rT-M386. Both lipases exhibited optimal activity and stability at pH 8. The G210C showed the highest stability in the presence of polar organic solvents at 50 °C compared to the rT-M386. Denatured protein analysis presented a significant change in the molecular ellipticity value above 60 °C, which verified the experimental result on the temperature and thermostability profile of G210C.

Phosphate and arsenate removal efficiency by thermostable ferritin enzyme from Pyrococcus furiosus using radioisotopes

KAUST Repository

Sevcenco, Ana-Maria

2015-03-13

Oxo-anion binding properties of the thermostable enzyme ferritin from Pyrococcus furiosus were characterized with radiography. Radioisotopes 32P and 76As present as oxoanions were used to measure the extent and the rate of their absorption by the ferritin. Thermostable ferritin proved to be an excellent system for rapid phosphate and arsenate removal from aqueous solutions down to residual concentrations at the picomolar level. These very low concentrations make thermostable ferritin a potential tool to considerably mitigate industrial biofouling by phosphate limitation or to remove arsenate from drinking water.

Characterization of mutants expressing thermostable D1 and D2 polypeptides of photosystem II in the cyanobacterium Synechococcus elongatus PCC 7942.

Science.gov (United States)

Haraguchi, Norihisa; Kaseda, Jun; Nakayama, Yasumune; Nagahama, Kazuhiro; Ogawa, Takahira; Matsuoka, Masayoshi

2018-06-08

Photosystem II complex embedded in thylakoid membrane performs oxygenic photosynthesis where the reaction center D1/D2 heterodimer accommodates all components of the electron transport chain. To express thermostable D1/D2 heterodimer in a cyanobacterium Synechococcus elongatus PCC 7942, we constructed a series of mutant strains whose psbA1 and psbD1 genes encoding, respectively, the most highly expressed D1 and D2 polypeptides were replaced with those of a thermophilic strain, Thermosynechococcus vulcanus. Because the C-terminal 16 amino acid sequences of D1 polypeptides should be processed prior to maturation but diverge from each other, we also constructed the psbA1ΔC-replaced strain expressing a thermostable D1 polypeptide devoid of the C-terminal extension. The psbA1/psbD1-replaced strain showed decreased growth rate and oxygen evolution rate, suggesting inefficient photosystem II. Immunoblot analyses for thermostable D1, D2 polypeptides revealed that the heterologous D1 protein was absent in thylakoid membrane from any mutant strains with psbA1, psbA1ΔC, and psbA1/psbD1-replacements, whereas the heterologous D2 protein was present in thylakoid membrane as well as purified photosystem II complex from the psbA1/psbD1-replaced strain. In the latter strain, the compensatory expression of psbA3 and psbD2 genes was elevated. These data suggest that heterologous D2 polypeptide could be combined with the host D1 polypeptide to form chimeric D1/D2 heterodimer, whereas heterologous D1 polypeptide even without the C-terminal extension was unable to make complex with the host D2 polypeptide. Since the heterologous D1 could not be detected even in the whole cells of psbA1/psbD1-replaced strain, the rapid degradation of unprocessed or unassembled heterologous D1 was implicated. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Crystallization and preliminary X-ray characterization of two thermostable DNA nucleases

International Nuclear Information System (INIS)

Kuettner, E. Bartholomeus; Pfeifer, Sven; Keim, Antje; Greiner-Stöffele, Thomas; Sträter, Norbert

2006-01-01

Two thermostable DNA nucleases from archaea were crystallized in different space groups; the crystals were suitable for X-ray analysis. Temperature-tolerant organisms are an important source to enhance the stability of enzymes used in biotechnological processes. The DNA-cleaving enzyme exonuclease III from Escherichia coli is used in several applications in gene technology. A thermostable variant could expand the applicability of the enzyme in these methods. Two homologous nucleases from Archaeoglobus fulgidus (ExoAf) and Methanothermobacter thermoautrophicus (ExoMt) were studied for this purpose. Both enzymes were crystallized in different space groups using (poly)ethylene glycols, 2,4-methyl pentandiol, dioxane, ethanol or 2-propanol as precipitants. The addition of a 10-mer DNA oligonucleotide was important to obtain monoclinic crystals of ExoAf and ExoMt that diffracted to resolutions better than 2 Å using synchrotron radiation. The crystal structures of the homologous proteins can serve as templates for genetic engineering of the E. coli exonuclease III and will aid in understanding the different catalytic properties of the enzymes

The Vaccine and Cervical Cancer Screen project 2 (VACCS 2 ...

African Journals Online (AJOL)

The Vaccine and Cervical Cancer Screen project 2 (VACCS 2): Linking cervical cancer screening to a two-dose HPV vaccination ... In VACCS 1 the feasibility of linking cervical cancer with HPV vaccination was demonstrated. ... Article Metrics.

Thermostable crude endoglucanase produced by Aspergillus ...

African Journals Online (AJOL)

Cellulases are used in many industries worldwide and there is an ever increasing need to isolate, produce or develop thermostable cellulases. Manipulation of fermentation techniques in order to obtain desirable product(s) can be one line of action. In this study Aspergillus fumigatus was grown on chopped wheat straw in a ...

Comparison of the thermostability of cellulases from various thermophilic fungi

Energy Technology Data Exchange (ETDEWEB)

Wojtczak, G; Breuil, C; Yamada, J; Saddler, J N

1987-10-01

The cellulase activities of six thermophilic fungi were compared. Although the thermophilic fungi grew at relatively high temperatures (> 45/sup 0/C) the optimum temperatures for assaying the various cellulase activities were only slightly higher than the optimum temperatures for the mesophilic fungi, Trichoderma harzianum. Over prolonged incubation (> 24 h) the thermophilic strains demonstrated a higher hydrolytic potential as a result of the greater thermostability of the cellulase components. Although the extracellular cellulase activities had similar pH and temperature optima, in some cases the thermostability of the extracellular components were considerably lower.

Canine Parvovirus (CPV) Vaccination: Comparison of Neutralizing Antibody Responses in Pups after Inoculation with CPV2 or CPV2b Modified Live Virus Vaccine

Science.gov (United States)

Pratelli, Annamaria; Cavalli, Alessandra; Martella, Vito; Tempesta, Maria; Decaro, Nicola; Carmichael, Leland Eugene; Buonavoglia, Canio

2001-01-01

Canine parvovirus type 2 (CPV2) emerged in 1978 as causative agent of a new disease of dogs. New antigenic variants (biotypes), designated CPV2a and CPV2b, became widespread during 1979 to 1980 and 1984, respectively. At the present time the original CPV2 has disappeared in the dog population and has been replaced by the two new viruses. In the present study the comparison of neutralizing antibody titers in two groups of pups (18 pups in each group) inoculated with CPV2 and CPV2b modified live virus vaccines is reported. Using the hemagglutination inhibition (HI) test, relevant differences between antibody titers, against either the homologous or the heterologous virus, were not constantly observed. Using the neutralization (Nt) test, however, the pups inoculated with CPV2 had antibody titers which were approximately 30 times higher to the homologous virus (mean, 4,732) than to the heterologous virus (CPV2b) (mean, 162). The results of these experiments support two conclusions: (i) the HI test may not always accurately evaluate the true immune status of dogs with respect to CPV, and (ii) dogs inoculated with CPV2 vaccine develop relatively low Nt antibody titers against the heterologous virus (CPV2b). These data may suggest an advantage for new vaccines, considering that most presently licensed vaccines are produced with CPV2, which no longer exists in the dog population. PMID:11329467

Canine parvovirus (CPV) vaccination: comparison of neutralizing antibody responses in pups after inoculation with CPV2 or CPV2b modified live virus vaccine.

Science.gov (United States)

Pratelli, A; Cavalli, A; Martella, V; Tempesta, M; Decaro, N; Carmichael, L E; Buonavoglia, C

2001-05-01

Canine parvovirus type 2 (CPV2) emerged in 1978 as causative agent of a new disease of dogs. New antigenic variants (biotypes), designated CPV2a and CPV2b, became widespread during 1979 to 1980 and 1984, respectively. At the present time the original CPV2 has disappeared in the dog population and has been replaced by the two new viruses. In the present study the comparison of neutralizing antibody titers in two groups of pups (18 pups in each group) inoculated with CPV2 and CPV2b modified live virus vaccines is reported. Using the hemagglutination inhibition (HI) test, relevant differences between antibody titers, against either the homologous or the heterologous virus, were not constantly observed. Using the neutralization (Nt) test, however, the pups inoculated with CPV2 had antibody titers which were approximately 30 times higher to the homologous virus (mean, 4,732) than to the heterologous virus (CPV2b) (mean, 162). The results of these experiments support two conclusions: (i) the HI test may not always accurately evaluate the true immune status of dogs with respect to CPV, and (ii) dogs inoculated with CPV2 vaccine develop relatively low Nt antibody titers against the heterologous virus (CPV2b). These data may suggest an advantage for new vaccines, considering that most presently licensed vaccines are produced with CPV2, which no longer exists in the dog population.

Design of thermostable rhamnogalacturonan lyase mutants from Bacillus licheniformis by combination of targeted single point mutations

DEFF Research Database (Denmark)

da Silva, Ines Isabel Cardoso Rodrigues; Jers, Carsten; Otten, Harm

2014-01-01

Rhamnogalacturonan I lyases (RGI lyases) (EC 4.2.2.-) catalyze cleavage of α-1,4 bonds between rhamnose and galacturonic acid in the backbone of pectins by β-elimination. In the present study, targeted improvement of the thermostability of a PL family 11 RGI lyase from Bacillus licheniformis (DSM......, were obtained due to additive stabilizing effects of single amino acid mutations (E434L, G55V, and G326E) compared to the wild type. The crystal structure of the B. licheniformis wild-type RGI lyase was also determined; the structural analysis corroborated that especially mutation of charged amino...

Improving the thermostability by introduction of arginines on the surface of α-L-rhamnosidase (r-Rha1) from Aspergillus niger.

Science.gov (United States)

Li, Lijun; Liao, Hui; Yang, Yan; Gong, Jianye; Liu, Jianan; Jiang, Zedong; Zhu, Yanbing; Xiao, Anfeng; Ni, Hui

2018-06-01

To improve the thermostability of α-L-rhamnosidase (r-Rha1), an enzyme previously identified from Aspergillus niger JMU-TS528, multiple arginine (Arg) residues were introduced into the r-Rha1 sequence to replace several lysine (Lys) residues that located on the surface of the folded r-Rha1. Hinted by in silico analysis, five surface Lys residues (K134, K228, K406, K440, K573) were targeted to produce a list of 5 single-residue mutants and 4 multiple-residue mutants using site-directed mutagenesis. Among these mutants, a double Lys to Arg mutant, i.e. K406R/K573R, showed the best thermostability improvement. The half-life of this mutant's enzyme activity increased 3 h at 60 °C, 23 min at 65 °C, and 3.5 min at 70 °C, when compared with the wild type. The simulated protein structure based interaction analysis and molecular dynamics calculation indicate that the thermostability improvement of the mutant K406R-K573R was possibly due to the extra hydrogen bonds, the additional cation-π interactions, and the relatively compact conformation. With the enhanced thermostability, the α-L-rhamnosidase mutant, K406R-K573R, has potentially broadened the r-Rha1 applications in food processing industry. Copyright © 2018 Elsevier B.V. All rights reserved.

A Design of Experiment approach to predict product and process parameters for a spray dried influenza vaccine.

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Kanojia, Gaurav; Willems, Geert-Jan; Frijlink, Henderik W; Kersten, Gideon F A; Soema, Peter C; Amorij, Jean-Pierre

2016-09-25

Spray dried vaccine formulations might be an alternative to traditional lyophilized vaccines. Compared to lyophilization, spray drying is a fast and cheap process extensively used for drying biologicals. The current study provides an approach that utilizes Design of Experiments for spray drying process to stabilize whole inactivated influenza virus (WIV) vaccine. The approach included systematically screening and optimizing the spray drying process variables, determining the desired process parameters and predicting product quality parameters. The process parameters inlet air temperature, nozzle gas flow rate and feed flow rate and their effect on WIV vaccine powder characteristics such as particle size, residual moisture content (RMC) and powder yield were investigated. Vaccine powders with a broad range of physical characteristics (RMC 1.2-4.9%, particle size 2.4-8.5μm and powder yield 42-82%) were obtained. WIV showed no significant loss in antigenicity as revealed by hemagglutination test. Furthermore, descriptive models generated by DoE software could be used to determine and select (set) spray drying process parameter. This was used to generate a dried WIV powder with predefined (predicted) characteristics. Moreover, the spray dried vaccine powders retained their antigenic stability even after storage for 3 months at 60°C. The approach used here enabled the generation of a thermostable, antigenic WIV vaccine powder with desired physical characteristics that could be potentially used for pulmonary administration. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Safety and immunogenicity of live-attenuated Japanese encephalitis SA 14-14-2 vaccine co-administered with measles vaccine in 9-month-old infants in Sri Lanka.

Science.gov (United States)

Wijesinghe, Pushpa Ranjan; Abeysinghe, M R Nihal; Yoksan, Sutee; Yao, Yafu; Zhou, Benli; Zhang, Lei; Yaich, Mansour; Neuzil, Kathleen M; Victor, John C

2014-08-20

To facilitate introduction of live attenuated SA 14-14-2 Japanese encephalitis vaccine (LJEV) into the National Immunization Programme of Sri Lanka, we evaluated the safety and immunogenicity of co-administration of LJEV and measles vaccine at 9 months of age. Serum immune responses were evaluated post-vaccination on days 28, 180, and 365 using JE neutralization test and anti-measles IgG ELISA. 278 infants received one dose of LJEV and measles vaccine. Of these, 257 were eligible for the per-protocol analysis. On Day 0, 14 infants (5.5%) were seropositive for JE, but none were seropositive for measles. At Day 28, seropositivity rates were 90.7% (95% CI, 86.4-93.9%) for JE and 84.8% (95% CI, 79.8-89.0%) for measles. The geometric mean titer for JE neutralizing antibodies was 111 (95% CI, 90-135), and the geometric mean concentration (GMC) for anti-measles IgG was 375 mI U/mL (95% CI, 351-400 mI U/mL). Over the next year, JE neutralizing antibody responses declined only slightly, with seropositivity at 87.4% (95% CI, 82.6-91.2%) at Day 365. In contrast, measles antibody levels continued to increase over time. Seropositivity for anti-measles IgG reached 97.2% (95% CI, 94.4-98.9%) at Day 365, and the GMC rose to 1202 mI U/mL (95% CI, 1077-1341 mI U/mL). Co-administration of LJEV and measles vaccine was also safe. Most adverse reactions were mild, and no serious adverse events were related to study vaccinations. The safety and immunogenicity of LJEV co-administered with measles vaccine in Sri Lankan infants is similar to that seen in other populations, and our results support use of LJEV at 9 months of age. Live SA 14-14-2 vaccine is now prequalified by the WHO for use in infants in Asia, and other countries may wish to introduce LJEV to combat this devastating disease. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Development of fowl cholera vaccine: I. Protection of Pasteurella multocida local isolate vaccine against challenge of homologous and heterologous strains.

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Supar

2001-03-01

Full Text Available Pasteurella multocida locally isolated from chicken and ducks (BCC 299, BCC 2331, DY1, DY2, 12TG, 15TG andimported strains (BCC 1359, 1362; HEDDLESTON group 1 and 6 respectively had been tested for its pathogenicity in theprevious study. The aims of this experiment were to study the preparation of local isolate pasteurellosis vaccines and to determine the protective effect of that vaccines in chicken against the highly pathogenic local isolates of P. multocida. Killed monovalent, bivalent and polyvalent pasteurellosis vaccines were prepared and each was adjunvanted with aluminum hydroxide gel at a final concentration of 1.5% and the cell concentration was equal to the No 10 of MacFarland tube standard. Each of the vaccine prepared was used to vaccinated on a group of six week old of layer chicken (8 per group. Each chicken was subcutaneously injected with 0.2 ml of vaccine, four weeks later each was boostered with similar vaccine with the same dose. Two weeks after giving the boostered vaccine each group of chicken were challenged, half with life bacterium of P. Multocida BCC 2331 and other with DY2. Any chick which survive after challenge was designated as protected by vaccination. Before vaccination 1 ml of blood was drawn from each of chicken and then two weeks apart up to challenge. Serum from each sample was separated and kept in deep freeze until tested by enzyme-linked immunosorbent assay (ELISA. Chicken vaccinated with killed whole cell P. multocida vaccines of monovalent (BCC 2331 or DY2 and bivalent (BCC 2331 + DY2 were protected against challenge with live bacterium of either BCC 2331 or DY2 at rate 67-100%. There was no protection in chicken vaccinated with either BCC 299, DY1, 12TG, 15TG, BCC 1359, or 1362 killed vaccine. Similarly no protection of chicken vaccinated with either DY1 + BCC299, 12TG + 15TG or BCC 1359 + BCC 1362 bivalent vaccines. The protection rate of the polyvalent local isolate vaccine was at average 50-75%. All

EFFECTS OF CHANGING THE INTERACTION BETWEEN SUBDOMAINS ON THE THERMOSTABILITY OF BACILLUS NEUTRAL PROTEASES

NARCIS (Netherlands)

EIJSINK, VGH; VRIEND, G; VANDERVINNE, B; HAZES, B; VANDENBURG, B; VENEMA, G

1992-01-01

Variants of the thermolabile neutral protease (Npr) of B. subtilis (Npr-sub) and the thermostable neutral protease of B. stearothermophilus (Npr-ste) were produced by means of site-directed mutagenesis and the effects of the mutations on thermostability were determined. Mutations were designed to

Thermostability in endoglucanases is fold-specific

Science.gov (United States)

2011-01-01

Background Endoglucanases are usually considered to be synergistically involved in the initial stages of cellulose breakdown-an essential step in the bioprocessing of lignocellulosic plant materials into bioethanol. Despite their economic importance, we currently lack a basic understanding of how some endoglucanases can sustain their ability to function at elevated temperatures required for bioprocessing, while others cannot. In this study, we present a detailed comparative analysis of both thermophilic and mesophilic endoglucanases in order to gain insights into origins of thermostability. We analyzed the sequences and structures for sets of endoglucanase proteins drawn from the Carbohydrate-Active enZymes (CAZy) database. Results Our results demonstrate that thermophilic endoglucanases and their mesophilic counterparts differ significantly in their amino acid compositions. Strikingly, these compositional differences are specific to protein folds and enzyme families, and lead to differences in intramolecular interactions in a fold-dependent fashion. Conclusions Here, we provide fold-specific guidelines to control thermostability in endoglucanases that will aid in making production of biofuels from plant biomass more efficient. PMID:21291533

Thermostability in endoglucanases is fold-specific

Directory of Open Access Journals (Sweden)

Wolt Jeffrey D

2011-02-01

Full Text Available Abstract Background Endoglucanases are usually considered to be synergistically involved in the initial stages of cellulose breakdown-an essential step in the bioprocessing of lignocellulosic plant materials into bioethanol. Despite their economic importance, we currently lack a basic understanding of how some endoglucanases can sustain their ability to function at elevated temperatures required for bioprocessing, while others cannot. In this study, we present a detailed comparative analysis of both thermophilic and mesophilic endoglucanases in order to gain insights into origins of thermostability. We analyzed the sequences and structures for sets of endoglucanase proteins drawn from the Carbohydrate-Active enZymes (CAZy database. Results Our results demonstrate that thermophilic endoglucanases and their mesophilic counterparts differ significantly in their amino acid compositions. Strikingly, these compositional differences are specific to protein folds and enzyme families, and lead to differences in intramolecular interactions in a fold-dependent fashion. Conclusions Here, we provide fold-specific guidelines to control thermostability in endoglucanases that will aid in making production of biofuels from plant biomass more efficient.

[Caprine arthritis-encephalitis: trial of an adjuvant vaccine preparation. I. Clinical and virological study].

Science.gov (United States)

Russo, P; Vitu, C; Fontaine, J J; Vignoni, M

1993-04-01

In purpose to protect goats against caprine arthritis encephalitis virus (CAEV), the first group of kids (I) was inoculated with purified, inactivated and adjuvant-treated virions, the second group (II) with adjuvant and the third one (III) with culture medium. 2-4 months later, the three groups were challenged with virulent CAEV by intraarticular route. On the clinical level, vaccinated and challenged kids show more early and severe arthritis than other groups. On the virological level, isolation of lentivirus from white blood cells and different organs is more important in group I than groups II and III. Therefore, vaccinations with inactivated and adjuvant-treated virions do not protect against a virulent challenge; there is an enhancement of lesions. We note that the adjuvant elicits a mild non-specific protection against virulent challenge.

Molecular characterization of a novel thermostable laccase PPLCC2 from the brown rot fungus Postia placenta MAD-698-R

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Hongde An

2015-11-01

Conclusions: This is the first identified thermo activated and thermostable laccase in brown rot fungi. This investigation will contribute to understanding the roles played by laccases in brown rot fungi.

Purification and Characterization of Thermostable Cellulase from ...

African Journals Online (AJOL)

The thermostable CMCase was purified with ion-exchange and gel filtration chromatography. Results: ... Conclusion: Due to its high temperature stability, the purified XM70-CMCase may be useful for industrial application such as biofuel, animal feed industry, paper industry and clarification of fruit juices. Keywords: ...

Interaction of porcine circovirus type 2 and Mycoplasma hyopneumoniae vaccines on dually infected pigs.

Science.gov (United States)

Seo, Hwi Won; Park, Su-Jin; Park, Changhoon; Chae, Chanhee

2014-05-01

The objective of this study was to determine the effects of porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae vaccinations on disease severity in an experimental PCV2-M. hyopneumoniae dual challenge model. Vaccine effectiveness was evaluated using microbiological (PCV2 viremia and M. hyopneumoniae nasal shedding), immunological (neutralizing antibodies and interferon-γ-secreting cells), and pathological (gross lung lesions, histopathologic pulmonary and lymphoid lesions, and the presence of PCV2 antigen and M. hyopneumoniae DNA within the lesions) evaluations. Although M. hyopneumoniae potentiates the severity of PCV2-associated lesions and lesion-associated PCV2 antigen in dually challenged pigs, vaccination against M. hyopneumoniae alone did not reduce PCV2 viremia, PCV2-induced lesions, or PCV2 antigen in dually challenged pigs. In addition, vaccination against PCV2 did not reduce the nasal shedding of M. hyopneumoniae, the M. hyopneumoniae-induced pulmonary lesions or the lesion-associated M. hyopneumoniae DNA in dually challenged pigs. Dual challenge with PCV2 and M. hyopneumoniae did not interfere with the induction of active immunity induced by a previous single vaccination for either PCV2 or M. hyopneumoniae. The results of this study demonstrated that (i) vaccination against M. hyopneumoniae alone did not decrease the potentiation of PCV2-induced lesions by M. hyopneumoniae and (ii) vaccination against PCV2 alone decreased the potentiation of PCV2-induced lesions by M. hyopneumoniae in dually challenged pigs. Copyright © 2014 Elsevier Ltd. All rights reserved.

Adenovirus-based vaccine against <i>Listeria monocytogenesi>

DEFF Research Database (Denmark)

Jensen, Søren; Steffensen, Maria Abildgaard; Jensen, Benjamin Anderschou Holbech

2013-01-01

The use of replication-deficient adenoviruses as vehicles for transfer of foreign genes offers many advantages in a vaccine setting, eliciting strong cellular immune responses involving both CD8(+) and CD4(+) T cells. Further improving the immunogenicity, tethering of the inserted target Ag to MHC...... linked to Ii compared with vaccination with the unlinked vaccine. Studies using knockout mice demonstrated that CD8(+) T cells were largely responsible for this protection, which is mediated through perforin-dependent lysis of infected cells and IFN-γ production. Taking the concept a step further...

Experiements with an inactivated hepatitis leptospirosis vaccine in vaccination programmes for dogs.

Science.gov (United States)

Wilson, J H; Hermann-Dekkers, W M; Leemans-Dessy, S; Meijer, J W

1977-06-25

A fluid adjuvanted vaccine consisting of inactivated hepatitis virus (iH) and leptospirae antigens (L) was developed. The vaccine (Kavak iHL; Duphar) was tested in several vaccination programmes both alone and in combination with freeze dried measles (M) or distemper (D) vaccines. The results demonstrate that this new vaccine is also effective in pups with maternally derived antibodies, although a second vaccination at 14 weeks of age is recommended to boost the first vaccination. For the booster vaccination either the iHL-vaccine or the liver attenuated hepatitis vaccine (H) can be used.

Evaluation of scanning 2D barcoded vaccines to improve data accuracy of vaccines administered.

Science.gov (United States)

Daily, Ashley; Kennedy, Erin D; Fierro, Leslie A; Reed, Jenica Huddleston; Greene, Michael; Williams, Warren W; Evanson, Heather V; Cox, Regina; Koeppl, Patrick; Gerlach, Ken

2016-11-11

Accurately recording vaccine lot number, expiration date, and product identifiers, in patient records is an important step in improving supply chain management and patient safety in the event of a recall. These data are being encoded on two-dimensional (2D) barcodes on most vaccine vials and syringes. Using electronic vaccine administration records, we evaluated the accuracy of lot number and expiration date entered using 2D barcode scanning compared to traditional manual or drop-down list entry methods. We analyzed 128,573 electronic records of vaccines administered at 32 facilities. We compared the accuracy of records entered using 2D barcode scanning with those entered using traditional methods using chi-square tests and multilevel logistic regression. When 2D barcodes were scanned, lot number data accuracy was 1.8 percentage points higher (94.3-96.1%, Pmanufacturer, month vaccine was administered, and vaccine type were associated with variation in accuracy for both lot number and expiration date. Two-dimensional barcode scanning shows promise for improving data accuracy of vaccine lot number and expiration date records. Adapting systems to further integrate with 2D barcoding could help increase adoption of 2D barcode scanning technology. Published by Elsevier Ltd.

A thermostable serralysin inhibitor from marine bacterium Flavobacterium sp. YS-80-122

Science.gov (United States)

Liang, Pengjuan; Li, Shangyong; Wang, Kun; Wang, Fang; Xing, Mengxin; Hao, Jianhua; Sun, Mi

2018-03-01

Serralysin inhibitors have been proposed as potent drugs against many diseases and may help to prevent further development of antibiotic-resistant pathogenic bacteria. In this study, a novel serralysin inhibitor gene, lupI, was cloned from the marine bacterium Flavobacterium sp. YS-80-122 and expressed in Escherichia coli. The deduced serralysin inhibitor, LupI, shows <40% amino acid identity to other reported serralysin inhibitors. Multiple sequence alignment and phylogenetic analysis of LupI with other serralysin inhibitors indicated that LupI was a novel type of serralysin inhibitor. The inhibitory constant for LupI towards its target metalloprotease was 0.64 μmol/L. LupI was thermostable at high temperature, in which 35.6%-90.7% of its inhibitory activity was recovered after treatment at 100°C for 1-60 min followed by incubation at 0°C. This novel inhibitor may represent a candidate drug for the treatment of serralysin-related infections.

Assessment of pulmonary antibodies with induced sputum and bronchoalveolar lavage induced by nasal vaccination against Pseudomonas aeruginosa: a clinical phase I/II study

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Freihorst Joachim

2007-08-01

Full Text Available Abstract Background Vaccination against Pseudomonas aeruginosa is a desirable albeit challenging strategy for prevention of airway infection in patients with cystic fibrosis. We assessed the immunogenicity of a nasal vaccine based on the outer membrane proteins F and I from Pseudomonas aeruginosa in the lower airways in a phase I/II clinical trial. Methods N = 12 healthy volunteers received 2 nasal vaccinations with an OprF-OprI gel as a primary and a systemic (n = 6 or a nasal booster vaccination (n = 6. Antibodies were assessed in induced sputum (IS, bronchoalveolar lavage (BAL, and in serum. Results OprF-OprI-specific IgG and IgA antibodies were found in both BAL and IS at comparable rates, but differed in the predominant isotype. IgA antibodies in IS did not correlate to the respective serum levels. Pulmonary antibodies were detectable in all vaccinees even 1 year after the vaccination. The systemic booster group had higher IgG levels in serum. However, the nasal booster group had the better long-term response with bronchial antibodies of both isotypes. Conclusion The nasal OprF-OprI-vaccine induces a lasting antibody response at both, systemic and airway mucosal site. IS is a feasible method to non-invasively assess bronchial antibodies. A further optimization of the vaccination schedule is warranted.

New vaccine strategies against enterotoxigenic Escherichia coli: II: Enhanced systemic and secreted antibody responses against the CFA/I fimbriae by priming with DNA and boosting with a live recombinant Salmonella vaccine

Directory of Open Access Journals (Sweden)

M.O. Lásaro

1999-02-01

Full Text Available The induction of systemic (IgG and mucosal (IgA antibody responses against the colonization factor I antigen (CFA/I of enterotoxigenic Escherichia coli (ETEC was evaluated in mice primed with an intramuscularly delivered CFA/I-encoding DNA vaccine followed by two oral immunizations with a live recombinant Salmonella typhimurium vaccine strain expressing the ETEC antigen. The booster effect induced by the oral immunization was detected two weeks and one year after the administration of the DNA vaccine. The DNA-primed/Salmonella-boosted vaccination regime showed a synergistic effect on the induced CFA/I-specific systemic and secreted antibody levels which could not be attained by either immunization strategy alone. These results suggest that the combined use of DNA vaccines and recombinant Salmonella vaccine strains can be a useful immunization strategy against enteric pathogens.

Synergistic function of four novel thermostable glycoside hydrolases from a long-term enriched thermophilic methanogenic digester

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Meng eWang

2015-05-01

Full Text Available In biofuel production from lignocellulose, low thermostability and product inhibition strongly restrict the enzyme activities and production process. Application of multiple thermostable glycoside hydrolases, forming an enzyme cocktail, can result in a synergistic action and therefore improve production efficiency and reduce operational costs. Therefore, increasing enzyme thermostabilities and compatibility are important for the biofuel industry. In this study, we reported the screening, cloning and biochemical characterization of four novel thermostable lignocellulose hydrolases from a metagenomic library of a long-term dry thermophilic methanogenic digester community, which were highly compatible with optimal conditions and specific activities. The optimal temperatures of the four enzymes, β-xylosidase, xylanase, β-glucosidase, and cellulase ranged from 60°C to 75°C, and over 80% residual activities were observed after 2 h incubation at 50°C. Mixtures of these hydrolases retained high residual synergistic activities after incubation with cellulose, xylan, and steam-exploded corncob at 50°C for 72 h. In addition, about 55% dry weight of steam-exploded corncob was hydrolyzed to glucose and xylose by the synergistic action of the four enzymes at 50°C for 48 h. This work suggested that since different enzymes from a same ecosystem could be more compatible, screening enzymes from a long-term enriching community could be a favorable strategy.

Can thermostable vaccines help address cold-chain challenges? Results from stakeholder interviews in six low- and middle-income countries.

Science.gov (United States)

Kristensen, Debra D; Lorenson, Tina; Bartholomew, Kate; Villadiego, Shirley

2016-02-10

This study captures the perspectives of stakeholders at multiple levels of the vaccine supply chain regarding their assessment of challenges with storing vaccines within recommended temperature ranges and their perceptions on the benefits of having vaccines with improved stability, including the potential short-term storage and transport of vaccines in a controlled-temperature chain. Semi-structured interviews were undertaken with 158 immunization stakeholders in six countries. Interviewees included national decision-makers and advisors involved in vaccine purchasing decisions, national Expanded Programme on Immunization managers, and health and logistics personnel at national, subnational, and health facility levels. Challenges with both heat and freeze-exposure of vaccines were recognized in all countries, with heat-exposure being a greater concern. Conditions leading to freeze-exposure including ice build-up due to poor refrigerator performance and improper icepack conditioning were reported by 53% and 28% of participants, respectively. Respondents were interested in vaccine products with improved heat/freeze-stability characteristics. The majority of those involved in vaccine purchasing indicated they would be willing to pay a US$0.05 premium per dose for a freeze-stable pentavalent vaccine (68%) or a heat-stable rotavirus vaccine (59%), although most (53%) preferred not to pay the premium for a heat-stable pentavalent vaccine if the increased stability required changing from a liquid to a lyophilized product. Most respondents (73%) were also interested in vaccines labeled for short-term use in a controlled-temperature chain. The majority (115/158) recognized the flexibility this would provide during outreach or should cold-chain breaks occur. Respondents were also aware that possible confusion might arise and additional training would be required if handling conditions were changed for some, but not all vaccines. Participating immunization stakeholders

Safety and immunogenicity of a live attenuated mumps vaccine: a phase I clinical trial.

Science.gov (United States)

Liang, Yan; Ma, Jingchen; Li, Changgui; Chen, Yuguo; Liu, Longding; Liao, Yun; Zhang, Ying; Jiang, Li; Wang, Xuan-Yi; Che, Yanchun; Deng, Wei; Li, Hong; Cui, Xiaoyu; Ma, Na; Ding, Dong; Xie, Zhongping; Cui, Pingfang; Ji, Qiuyan; Wang, JingJing; Zhao, Yuliang; Wang, Junzhi; Li, Qihan

2014-01-01

Mumps, a communicable, acute and previously well-controlled disease, has had recent and occasional resurgences in some areas. A randomized, double-blind, controlled and multistep phase I study of an F-genotype attenuated mumps vaccine produced in human diploid cells was conducted. A total of 300 subjects were enrolled and divided into 4 age groups: 16-60 years, 5-16 years, 2-5 years and 8-24 months. The groups were immunized with one injection per subject. Three different doses of the F-genotype attenuated mumps vaccine, A (3.5 ± 0.25 logCCID50), B (4.25 ± 0.25 logCCID50) and C (5.0 ± 0.25 logCCID50), as well as a placebo control and a positive control of a licensed A-genotype vaccine (S79 strain) were used. The safety and immunogenicity of this vaccine were compared with those of the controls. The safety evaluation suggested that mild adverse reactions were observed in all groups. No serious adverse event (SAE) was reported throughout the trial. The immunogenicity test showed a similar seroconversion rate of the neutralizing and ELISA antibody in the 2- to 5-year-old and 8- to 24-month-old groups compared with the seroconversion rate in the positive control. The GMT of the neutralizing anti-F-genotype virus antibodies in the vaccine groups was slightly higher than that in the positive control group. The F-genotype attenuated mumps vaccine evaluated in this clinical trial was demonstrated to be safe and have effective immunogenicity vs. control.

Structure Based Thermostability Prediction Models for Protein Single Point Mutations with Machine Learning Tools.

Directory of Open Access Journals (Sweden)

Lei Jia

Full Text Available Thermostability issue of protein point mutations is a common occurrence in protein engineering. An application which predicts the thermostability of mutants can be helpful for guiding decision making process in protein design via mutagenesis. An in silico point mutation scanning method is frequently used to find "hot spots" in proteins for focused mutagenesis. ProTherm (http://gibk26.bio.kyutech.ac.jp/jouhou/Protherm/protherm.html is a public database that consists of thousands of protein mutants' experimentally measured thermostability. Two data sets based on two differently measured thermostability properties of protein single point mutations, namely the unfolding free energy change (ddG and melting temperature change (dTm were obtained from this database. Folding free energy change calculation from Rosetta, structural information of the point mutations as well as amino acid physical properties were obtained for building thermostability prediction models with informatics modeling tools. Five supervised machine learning methods (support vector machine, random forests, artificial neural network, naïve Bayes classifier, K nearest neighbor and partial least squares regression are used for building the prediction models. Binary and ternary classifications as well as regression models were built and evaluated. Data set redundancy and balancing, the reverse mutations technique, feature selection, and comparison to other published methods were discussed. Rosetta calculated folding free energy change ranked as the most influential features in all prediction models. Other descriptors also made significant contributions to increasing the accuracy of the prediction models.

Effect of pulsed electric field treatment on enzyme kinetics and thermostability of endogenous ascorbic acid oxidase in carrots (Daucus carota cv. Nantes).

Science.gov (United States)

Leong, Sze Ying; Oey, Indrawati

2014-03-01

The objective of this research was to study the enzyme kinetics and thermostability of endogenous ascorbic acid oxidase (AAO) in carrot purée (Daucus carota cv. Nantes) after being treated with pulsed electric field (PEF) processing. Various PEF treatments using electric field strength between 0.2 and 1.2kV/cm and pulsed electrical energy between 1 and 520kJ/kg were conducted. The enzyme kinetics and the kinetics of AAO thermal inactivation (55-70°C) were described using Michaelis-Menten model and first order reaction model, respectively. Overall, the estimated Vmax and KM values were situated in the same order of magnitude as the untreated carrot purée after being exposed to pulsed electrical energy between 1 and 400kJ/kg, but slightly changed at pulsed electrical energy above 500kJ/kg. However, AAO presented different thermostability depending on the electric field strength applied. After PEF treatment at the electric field strength between 0.2 and 0.5kV/cm, AAO became thermolabile (i.e. increase in inactivation rate (k value) at reference temperature) but the temperature dependence of k value (Ea value) for AAO inactivation in carrot purée decreased, indicating that the changes in k values were less temperature dependent. It is obvious that PEF treatment affects the temperature stability of endogenous AAO. The changes in enzyme kinetics and thermostability of AAO in carrot purée could be related to the resulting carrot purée composition, alteration in intracellular environment and the effective concentration of AAO released after being subjected to PEF treatment. Copyright © 2013 Elsevier Ltd. All rights reserved.

Production of thermostable and organic solvent-tolerant alkaline ...

African Journals Online (AJOL)

An alkaliphilic bacterium producing organic solvent-tolerant and thermostable alkaline protease was isolated from poultry litter site and identified as Bacillus coagulans PSB-07. Protease production under different submerged fermentation conditions were investigated with the aim of optimizing yield of enzyme. B. coagulans ...

Crystallization and preliminary X-ray diffraction studies of the precursor protein of a thermostable variant of papain

International Nuclear Information System (INIS)

Roy, Sumana; Choudhury, Debi; Chakrabarti, Chandana; Biswas, Sampa; Dattagupta, J. K.

2011-01-01

The crystallization of the precursor of a thermostable variant of papain and the collection of diffraction data to 2.6 Å resolution are reported. The crystallization of a recombinant thermostable variant of pro-papain has been carried out. The mutant pro-enzyme was expressed in Escherichia coli as inclusion bodies, refolded, purified and crystallized. The crystals belonged to space group P2 1 , with unit-cell parameters a = 42.9, b = 74.8, c = 116.5 Å, β = 93.0°, and diffracted to 2.6 Å resolution using synchrotron radiation. Assuming the presence of two molecules in the asymmetric unit, the calculated Matthews coefficient is 2.28 Å 3 Da −1 , corresponding to a solvent content of 46%. Initial attempts to solve the structure using molecular-replacement techniques were successful

Isolation and molecular characterization of thermostable phytase from Bacillus subtilis (BSPhyARRMK33).

Science.gov (United States)

Reddy, Chinreddy Subramanyam; Achary, V Mohan Murali; Manna, Mrinalini; Singh, Jitender; Kaul, Tanushri; Reddy, Malireddy K

2015-03-01

The thermostable phytase gene was isolated from Bacillus subtilis ARRMK33 (BsPhyARRMK33). The gene has an ORF of 1152 bp and that encodes a protein of 383 amino acids. Sequence analysis showed high homology with Bacillus sp. phytase proteins, but no similarity was found with other phytases. SDS-PAGE analysis exhibited a predicted molecular mass of 42 kDa. Homology modeling of BsPhyARRMK33 protein based on Bacillus amyloliquefaciens crystal structure disclosed its β-propeller structure. BsPhyARRMK33 recombinant plasmid in pET-28a(+) was expressed in Rosetta gami B DE3 cells and the maximum phytase activity 15.3 U mg(-1) obtained. The enzyme exhibits high thermostability at various temperatures and broad pH ranges. The recombinant protein retained 74% of its original activity after incubation at 95 °C for 10 min. In the presence of Ca(2+), the recombinant phytase activity was maximal where as it was inhibited by EDTA. The optimal pH and temperature for the recombinant phytase activity is achieved at 7.0 and 55 °C, respectively. Thermostable nature and wide range of pH are promising features of recombinant BsPhyARRMK33 protein that may be employed as an efficient alternative to commercially known phytases and thereby alleviate environmental eutrophication.

Bilirubin oxidase-like proteins from Podospora anserina: promising thermostable enzymes for application in transformation of plant biomass.

Science.gov (United States)

Xie, Ning; Ruprich-Robert, Gwenaël; Silar, Philippe; Chapeland-Leclerc, Florence

2015-03-01

Plant biomass degradation by fungi is a critical step for production of biofuels, and laccases are common ligninolytic enzymes envisioned for ligninolysis. Bilirubin oxidases (BODs)-like are related to laccases, but their roles during lignocellulose degradation have not yet been fully investigated. The two BODs of the ascomycete fungus Podospora anserina were characterized by targeted gene deletions. Enzymatic assay revealed that the bod1(Δ) and bod2(Δ) mutants lost partly a thermostable laccase activity. A triple mutant inactivated for bod1, bod2 and mco, a previously investigated multicopper oxidase gene distantly related to laccases, had no thermostable laccase activity. The pattern of fruiting body production in the bod1(Δ) bod2(Δ) double mutant was changed. The bod1(Δ) and bod2(Δ) mutants were reduced in their ability to grow on ligneous and cellulosic materials. Furthermore, bod1(Δ) and bod2(Δ) mutants were defective towards resistance to phenolic substrates and H2 O2 , which may also impact lignocellulose breakdown. Double and triple mutants were more affected than single mutants, evidencing redundancy of function among BODs and mco. Overall, the data show that bod1, bod2 and mco code for non-canonical thermostable laccases that participate in the degradation of lignocellulose. Thanks to their thermal stability, these enzymes may be more promising candidate for biotechnological application than canonical laccases. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

A novel non-thermostable deuterolysin from Aspergillus oryzae.

Science.gov (United States)

Maeda, Hiroshi; Katase, Toru; Sakai, Daisuke; Takeuchi, Michio; Kusumoto, Ken-Ichi; Amano, Hitoshi; Ishida, Hiroki; Abe, Keietsu; Yamagata, Youhei

2016-09-01

Three putative deuterolysin (EC 3.4.24.29) genes (deuA, deuB, and deuC) were found in the Aspergillus oryzae genome database ( http://www.bio.nite.go.jp/dogan/project/view/AO ). One of these genes, deuA, was corresponding to NpII gene, previously reported. DeuA and DeuB were overexpressed by recombinant A. oryzae and were purified. The degradation profiles against protein substrates of both enzymes were similar, but DeuB showed wider substrate specificity against peptidyl MCA-substrates compared with DeuA. Enzymatic profiles of DeuB except for thermostability also resembled those of DeuA. DeuB was inactivated by heat treatment above 80° C, different from thermostable DeuA. Transcription analysis in wild type A. oryzae showed only deuB was expressed in liquid culture, and the addition of the proteinous substrate upregulated the transcription. Furthermore, the NaNO3 addition seems to eliminate the effect of proteinous substrate for the transcription of deuB.

Structure of a thermostable serralysin from Serratia sp. FS14 at 1.1 Å resolution.

Science.gov (United States)

Wu, Dongxia; Ran, Tinting; Wang, Weiwu; Xu, Dongqing

2016-01-01

Serralysin is a well studied metalloprotease, and typical serralysins are not thermostable. The serralysin isolated from Serratia sp. FS14 was found to be thermostable, and in order to reveal the mechanism responsible for its thermostability, the crystal structure of serralysin from Serratia sp. FS14 was solved to a crystallographic R factor of 0.1619 at 1.10 Å resolution. Similar to its homologues, it mainly consists of two domains: an N-terminal catalytic domain and a `parallel β-roll' C-terminal domain. Comparative studies show that the shape of the catalytic active-site cavity is more open owing to the 189-198 loop, with a short 310-helix protruding further from the molecular surface, and that the β-sheets comprising the `parallel β-roll' are longer than those in its homologues. The formation of hydrogen bonds from one of the nonconserved residues (Asn200) to Lys27 may contribute to the thermostability.

A boosting skin vaccination with dissolving microneedle patch encapsulating M2e vaccine broadens the protective efficacy of conventional influenza vaccines.

Science.gov (United States)

Zhu, Wandi; Pewin, Winston; Wang, Chao; Luo, Yuan; Gonzalez, Gilbert X; Mohan, Teena; Prausnitz, Mark R; Wang, Bao-Zhong

2017-09-10

The biodegradable microneedle patch (MNP) is a novel technology for vaccine delivery that could improve the immunogenicity of vaccines. To broaden the protective efficiency of conventional influenza vaccines, a new 4M2e-tFliC fusion protein construct containing M2e sequences from different subtypes was generated. Purified fusion protein was encapsulate into MNPs with a biocompatible polymer for use as a boosting vaccine. The results demonstrated that mice receiving a conventional inactivated vaccine followed by a skin-applied dissolving 4M2e-tFliC MNP boost could better maintain the humoral antibody response than that by the conventional vaccine-prime alone. Compared with an intramuscular injection boost, mice receiving the MNP boost showed significantly enhanced cellular immune responses, hemagglutination-inhibition (HAI) titers, and neutralization titers. Increased frequency of antigen-specific plasma cells and long-lived bone marrow plasma cells was detected in the MNP boosted group as well, indicating that skin vaccination with 4M2e-tFliC facilitated a long-term antibody-mediated immunity. The 4M2e-tFliC MNP-boosted group also possessed enhanced protection against high lethal dose challenges against homologous A/PR/8/34 and A/Aichi/2/68 viruses and protection for a majority of immunized mice against a heterologous A/California/07/2009 H1N1 virus. High levels of M2e specific immune responses were observed in the 4M2e-tFliC MNP-boosted group as well. These results demonstrate that a skin-applied 4M2e-tFliC MNP boosting immunization to seasonal vaccine recipients may be a rapid approach for increasing the protective efficacy of seasonal vaccines in response to a significant drift seen in circulating viruses. The results also provide a new perspective for future exploration of universal influenza vaccines. Copyright © 2017 Elsevier B.V. All rights reserved.

Highly thermostable fluorescent proteins

Science.gov (United States)

Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

2011-03-22

Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

MVA E2 recombinant vaccine in the treatment of human papillomavirus infection in men presenting intraurethral flat condyloma: a phase I/II study.

Science.gov (United States)

Albarran Y Carvajal, Antonio; de la Garza, Alfonso; Cruz Quiroz, Benitez Jose Cecilio; Vazquez Zea, Eduardo; Díaz Estrada, Ismael; Mendez Fuentez, Ernesto; López Contreras, Mario; Andrade-Manzano, Alejandro; Padilla, Santiago; Varela, Axel Ramírez; Rosales, Ricardo

2007-01-01

Human papillomavirus (HPV) is the etiologic agent for warts and cervical cancer. In Mexico, the death rate from cervical cancer is extremely high, and statistical data show that since 1990 the number of deaths is increasing. Condylomas and cancer of the penis are the most common lesions presented in men; bladder and prostate cancer in men are also associated with the presence of HPV. Since HPV is transmitted by sexual intercourse, treating both partners is necessary in order to eliminate the virus in the population. Approaches to this include preventative vaccines such as Gardasil, and therapeutic vaccines to treat established infections in both men and women. This will be the only way to decrease the numbers of deaths due to this malignancy. We conducted a phase I/II clinical trial to evaluate the potential use of the recombinant vaccinia viral vaccine MVA E2 (composed of modified vaccinia virus Ankara [MVA] expressing the E2 gene of bovine papillomavirus) to treat flat condyloma lesions associated with oncogenic HPV in men. Fifty male patients with flat condyloma lesions were treated with either MVA E2 therapeutic vaccine or fluorouracil (5-fluorouracil). Thirty men received the therapeutic vaccine, at a total of 10(6) virus particles per dose, administered directly into the urethra once every week over a 4-week period. Twenty control patients were treated with 5% fluorouracil 1mL twice weekly over a 4-week period directly into the urethra. Reduction of lesions or absence of papillomavirus infection was monitored by colposcopy and histologic analysis. The immune response after MVA E2 treatment was determined by measuring the antibodies against the MVA E2 virus and by analyzing the lymphocyte cytotoxic activity against cancer cells bearing oncogenic papillomavirus. Presence of papillomavirus was determined by the Hybrid Capture method. Twenty-eight of 30 patients showed no lesion or presence of papillomavirus as diagnosed by colposcopy and brush histologic

Thermostable Alginate degrading enzymes and their methods of use

NARCIS (Netherlands)

Hreggvidsson, Gudmundur Oli; Jonsson, Oskar W.J.; Bjornsdottir, Bryndis; Fridjonsson, Hedinn O; Altenbuchner, Josef; Watzlawick, Hildegard; Dobruchowska, Justyna; Kamerling, Johannis

2015-01-01

The present invention relates to the identification, production and use of thermostable alginate lyase enzymes that can be used to partially degrade alginate to yield oligosaccharides or to give complete degradation of alginate to yield (unsaturated) mono-uronates.

Better vaccines for healthier life. Part II. Conference report of the DCVMN International 14th Annual General Meeting Hanoi, Vietnam.

Science.gov (United States)

Pagliusi, Sonia; Tippoo, Patrick; Sivaramakrishnan, Venkatraman; Nguyen, Thuvan

2014-11-12

New vaccines are required to meet the public health challenges of the next generation and many unmet global health needs can be addressed by developing countries vaccine manufacturers such as lower-cost vaccines based on single-dose, thermostable formulations, efficacious in children with compromised gastrointestinal tracts. GMP compliance is also a challenge, as sometimes innovation and clinical development focus is not accompanied by command of scale-up and quality assurance for large volume manufacturing and supply. Identifying and addressing such challenges, beyond cost and cold-chain space, including safety considerations and health worker behavior, regulatory alliances and harmonization to foster access to vaccines, will help countries to ensure sustainable immunization. There needs to be continuous and close management of the global vaccine supply both at national and international levels, requiring careful risk management, coordination and cooperation with manufacturers. Successful partnership models based on sharing a common goal, mutual respect and good communication were discussed among stakeholders. Copyright © 2014. Published by Elsevier Ltd.. All rights reserved.

Vaccination-challenge studies with a Port Chalmers/73 (H3N2)-based swine influenza virus vaccine: Reflections on vaccine strain updates and on the vaccine potency test.

Science.gov (United States)

De Vleeschauwer, Annebel; Qiu, Yu; Van Reeth, Kristien

2015-05-11

The human A/Port Chalmers/1/73 (H3N2) influenza virus strain, the supposed ancestor of European H3N2 swine influenza viruses (SIVs), was used in most commercial SIV vaccines in Europe until recently. If manufacturers want to update vaccine strains, they have to perform laborious intratracheal (IT) challenge experiments and demonstrate reduced virus titres in the lungs of vaccinated pigs. We aimed to examine (a) the ability of a Port Chalmers/73-based commercial vaccine to induce cross-protection against a contemporary European H3N2 SIV and serologic cross-reaction against H3N2 SIVs from Europe and North America and (b) the validity of intranasal (IN) challenge and virus titrations of nasal swabs as alternatives for IT challenge and titrations of lung tissue in vaccine potency tests. Pigs were vaccinated with Suvaxyn Flu(®) and challenged by the IT or IN route with sw/Gent/172/08. Post-vaccination sera were examined in haemagglutination-inhibition assays against vaccine and challenge strains and additional H3N2 SIVs from Europe and North America, including an H3N2 variant virus. Tissues of the respiratory tract and nasal swabs were collected 3 days post challenge (DPCh) and from 0-7 DPCh, respectively, and examined by virus titration. Two vaccinations consistently induced cross-reactive antibodies against European H3N2 SIVs from 1998-2012, but minimal or undetectable antibody titres against North American viruses. Challenge virus titres in the lungs, trachea and nasal mucosa of the vaccinated pigs were significantly reduced after both IT and IN challenge. Yet the reduction of virus titres and nasal shedding was greater after IT challenge. The Port Chalmers/73-based vaccine still offered protection against a European H3N2 SIV isolated 35 years later and with only 86.9% amino acid homology in its HA1, but it is unlikely to protect against H3N2 SIVs that are endemic in North America. We use our data to reflect on vaccine strain updates and on the vaccine potency test

MHC-I-restricted epitopes conserved among variola and other related orthopoxviruses are recognized by T cells 30 years after vaccination

DEFF Research Database (Denmark)

Tang, Sheila Tuyet; Wang, M.; Lamberth, K.

2008-01-01

It is many years since the general population has been vaccinated against smallpox virus. Here, we report that human leukocyte antigen (HLA) class I restricted T cell epitopes can be recognized more than 30 years after vaccination. Using bioinformatic methods, we predicted 177 potential cytotoxic T...... lymphocyte epitopes. Eight epitopes were confirmed to stimulate IFN-gamma release by T cells in smallpox-vaccinated subjects. The epitopes were restricted by five supertypes (HLA-A1, -A2, -A24 -A26 and -B44). Significant T cell responses were detected against 8 of 45 peptides with an HLA class I affinity...... of K(D) less than or equal to 5 nM, whereas no T cell responses were detected against 60 peptides with an HLA affinity of K(D) more than 5 nM. All epitopes were fully conserved in seven variola, vaccinia and cowpox strains. Knowledge of the long-term response to smallpox vaccination may lead...

Tyr51: Key Determinant of the Low Thermostability of the Colwellia psychrerythraea Cold-Shock Protein.

Science.gov (United States)

Lee, Yeongjoon; Kwak, Chulhee; Jeong, Ki-Woong; Durai, Prasannavenkatesh; Ryu, Kyoung-Seok; Kim, Eun-Hee; Cheong, Chaejoon; Ahn, Hee-Chul; Kim, Hak Jun; Kim, Yangmee

2018-05-18

Cold-shock proteins (Csps) are expressed at lower-than-optimum temperatures, and they function as RNA chaperones; however, no structural studies on psychrophilic Csps have been reported. Here, we aimed to investigate the structure and dynamics of the Csp of psychrophile Colwellia psychrerythraea 34H, ( Cp-Csp). Although Cp-Csp shares sequence homology, common folding patterns, and motifs, including a five β-stranded barrel, with its thermophilic counterparts, its thermostability (37 °C) was markedly lower than those of other Csps. Cp-Csp binds heptathymidine with an affinity of 10 -7 M, thereby increasing its thermostability to 50 °C. Nuclear magnetic resonance spectroscopic analysis of the Cp-Csp structure and backbone dynamics revealed a flexible structure with only one salt bridge and 10 residues in the hydrophobic cavity. Notably, Cp-Csp contains Tyr51 instead of the conserved Phe in the hydrophobic core, and its phenolic hydroxyl group projects toward the surface. The Y51F mutation increased the stability of hydrophobic packing and may have allowed for the formation of a K3-E21 salt bridge, thereby increasing its thermostability to 43 °C. Cp-Csp exhibited conformational exchanges in its ribonucleoprotein motifs 1 and 2 (754 and 642 s -1 ), and heptathymidine binding markedly decreased these motions. Cp-Csp lacks salt bridges and has longer flexible loops and a less compact hydrophobic cavity resulting from Tyr51 compared to mesophilic and thermophilic Csps. These might explain the low thermostability of Cp-Csp. The conformational flexibility of Cp-Csp facilitates its accommodation of nucleic acids at low temperatures in polar oceans and its function as an RNA chaperone for cold adaptation.

Clinical, serological and echocardiographic examination of healthy field dogs before and after vaccination with a commercial tetravalent leptospirosis vaccine.

Science.gov (United States)

Spiri, Andrea M; Rodriguez-Campos, Sabrina; Matos, José M; Glaus, Tony M; Riond, Barbara; Reusch, Claudia E; Hofmann-Lehmann, Regina; Willi, Barbara

2017-05-25

Leptospirosis is a re-emerging bacterial zoonosis caused by spirochetes of the genus Leptospira. Severe disease has been reported in dogs in Europe despite vaccination with bivalent Leptospira vaccines. Recently, a tetravalent canine Leptospira vaccine (Nobivac® L4) was licenced in Europe. The goal of this study was to investigate clinical signs, microscopic agglutination test (MAT) titres, haematology, blood biochemistry, cardiac (c) Troponin I levels and echocardiography before and after vaccination with this tetravalent vaccine. Forty-eight healthy dogs were prospectively enrolled and vaccinated twice, 3-4 weeks apart (T0 and T1). Before vaccination (T0) and 16-31 days after the second vaccination (T2), MAT (n = 48), haematology (n = 48), blood biochemistry (n = 36) and cTroponin I measurements (n = 29) were performed, and MAT was repeated 347-413 days after the second vaccination (T3, n = 44). Echocardiography was performed before the first and second vaccination (T0 and T1, n = 24). Mild and transient clinical signs within 5 days following the first and second vaccination occurred in 23% and 10% of the dogs, respectively. Before the first vaccination (T0), all dogs showed negative MAT titres for the tested serovars except for Canicola (50% with titres 100-400). At T2, positive MAT titres to the serovars Canicola (100%), Australis (89%), Grippotyphosa (86%), Bratislava (60%), Autumnalis (58%), Copenhageni (42%), Pomona (12%), Pyrogenes (8%) and Icterohaemorrhagiae (2%) were found. Median to high titres (≥ 400) were most common to the serovar Canicola (92%) and less common to the serovars Australis (41%), Grippotyphosa (21%), Bratislava (12%), Autumnalis (4%), Pyrogenes (4%) and Pomona (2%). At T3, positive MAT titres (titre range: 100-400) were found in 2-18% of the dogs to serovars of the vaccine serogroups and in 2-18% of the dogs to the non-vaccine serovars Pomona, Autumnalis, Pyrogenes and Ballum. Haematology, blood biochemistry, c

Challenges in the rabbit haemorrhagic disease 2 (RHDV2) molecular diagnosis of vaccinated rabbits.

Science.gov (United States)

Carvalho, C L; Duarte, E L; Monteiro, M; Botelho, A; Albuquerque, T; Fevereiro, M; Henriques, A M; Barros, S S; Duarte, Margarida Dias

2017-01-01

Molecular methods are fundamental tools for the diagnosis of viral infections. While interpretation of results is straightforward for unvaccinated animals, where positivity represents ongoing or past infections, the presence of vaccine virus in the tissues of recently vaccinated animals may mislead diagnosis. In this study, we investigated the interference of RHDV2 vaccination in the results of a RT-qPCR for RHDV2 detection, and possible associations between mean Cq values of five animal groups differing in age, vaccination status and origin (domestic/wild). Viral sequences from vaccinated rabbits that died of RHDV2 infection (n=14) were compared with the sequences from the commercial vaccines used in those animals. Group Cq means were compared through Independent t-test and One-way ANOVA. We proved that RHDV2 vaccine-RNA is not detected by the RT-qPCR as early as 15days post-vaccination, an important fact in assisting results interpretation for diagnosis. Cq values of vaccinated and non-vaccinated infected domestic adults showed a statistically significant difference (pRHDV2-victimised rabbits. Although the reduced number of vaccinated young animals analysed hampered a robust statistical analysis, this occurrence suggests that passively acquired maternal antibodies may inhibit the active immune response to vaccination, delaying protection and favouring disease progression. Our finding emphasises the importance of adapting kitten RHDV2 vaccination schedules to circumvent this interference phenomenon. Copyright © 2016 Elsevier B.V. All rights reserved.

Immunogenicity and safety evaluation of bivalent types 1 and 3 oral poliovirus vaccine by comparing different poliomyelitis vaccination schedules in China: A randomized controlled non-inferiority clinical trial.

Science.gov (United States)

Qiu, Jingjun; Yang, Yunkai; Huang, Lirong; Wang, Ling; Jiang, Zhiwei; Gong, Jian; Wang, Wei; Wang, Hongyan; Guo, Shaohong; Li, Chanjuan; Wei, Shuyuan; Mo, Zhaojun; Xia, Jielai

2017-06-03

The type 2 component of the oral poliovirus vaccine is targeted for global withdrawal through a switch from the trivalent oral poliovirus vaccine (tOPV) to a bivalent oral poliovirus vaccine (bOPV). The switch is intended to prevent paralytic polio caused by circulating vaccine-derived poliovirus type 2. We aimed to assess the immunogenicity and safety profile of 6 vaccination schedules with different sequential doses of inactivated poliovirus vaccine (IPV), tOPV, or bOPV. A randomized controlled trial was conducted in China in 2015. Healthy newborn babies randomly received one of the following 6 vaccination schedules: cIPV-bOPV-bOPV(I-B-B), cIPV-tOPV-tOPV(I-T-T), cIPV-cIPV-bOPV(I-I-B), cIPV-cIPV-tOPV(I-I-T), cIPV-cIPV-cIPV(I-I-I), or tOPV-tOPV-tOPV(T-T-T). Doses were administered sequentially at 4-6 week intervals after collecting baseline blood samples. Patients were proactively followed up for observation of adverse events after the first dose and 30 days after all doses. The primary study objective was to investigate the immunogenicity and safety profile of different vaccine schedules, evaluated by seroconversion, seroprotection and antibody titer against poliovirus types 1, 2, and 3 in the per-protocol population. Of 600 newborn babies enrolled, 504 (84.0%) were included in the per-protocol population. For type 1 poliovirus, the differences in the seroconversion were 1.17% (95% CI = -2.74%, 5.08%) between I-B-B and I-T-T and 0.00% (95% CI: -6.99%, 6.99%) between I-I-B and I-I-T; for type 3 poliovirus, differences in the seroconversion were 3.49% (95% CI: -1.50%, 8.48%) between I-B-B and I-T-T and -2.32% (95% CI: -5.51%, 0.86%) between I-I-B and I-I-T. The non-inferiority conclusion was achieved in both poliovirus type 1 and 3 with the margin of -10%. Of 24 serious adverse events reported, no one was vaccine-related. The vaccination schedules with bOPV followed by one or 2 doses of IPV were recommended to substitute for vaccinations involving tOPV without

Immunogenicity and safety of tetravalent dengue vaccine in 2-11 year-olds previously vaccinated against yellow fever: randomized, controlled, phase II study in Piura, Peru.

Science.gov (United States)

Lanata, Claudio F; Andrade, Teresa; Gil, Ana I; Terrones, Cynthia; Valladolid, Omar; Zambrano, Betzana; Saville, Melanie; Crevat, Denis

2012-09-07

In a randomized, placebo-controlled, monocenter, observer blinded study conducted in an area where dengue is endemic, we assessed the safety and immunogenicity of a recombinant, live, attenuated, tetravalent dengue vaccine candidate (CYD-TDV) in 2-11 year-olds with varying levels of pre-existing yellow-fever immunity due to vaccination 1-7 years previously. 199 children received 3 injections of CYD-TDV (months 0, 6 and 12) and 99 received placebo (months 0 and 6) or pneumococcal polysaccharide vaccine (month 12). One month after the third dengue vaccination, serotype specific neutralizing antibody GMTs were in the range of 178-190 (1/dil) (versus 16.7-38.1 in the control group), a 10-20 fold-increase from baseline, and 94% of vaccines were seropositive to all four serotypes (versus 39% in the control group). There were no vaccine-related SAEs. The observed reactogenicity profile was consistent with phase I studies, with severity grade 1-2 injection site pain, headache, malaise and fever most frequently reported and no increase after subsequent vaccinations. Virologically confirmed dengue cases were seen after completion of the 3 doses: 1 in the CYD-TDV group (N=199), and 3 in the control group (N=99). A 3-dose regimen of CYD-TDV had a good safety profile in 2-11 year olds with a history of YF vaccination and elicited robust antibody responses that were balanced against the four serotypes. Copyright © 2012 Elsevier Ltd. All rights reserved.

Approaches for improving thermostability characteristics in cellulases.

Science.gov (United States)

Anbar, Michael; Bayer, Edward A

2012-01-01

Many efforts have been invested to reduce the cost of biofuel production to substitute renewable sources of energy for fossil-based fuels. At the forefront of these efforts are the initiatives to convert plant-derived cellulosic material to biofuels. Although significant improvements have been achieved recently in cellulase engineering in both efficiency and cost reduction, complete degradation of lignocellulosic material still requires very long periods of time and high enzyme loads. Thermostable cellulases offer many advantages in the bioconversion process, which include increase in specific activity, higher levels of stability, inhibition of microbial growth, increase in mass transfer rate due to lower fluid viscosity, and greater flexibility in the bioprocess. Besides rational design methods, which require deep understanding of protein structure-function relationship, two of the major methods for improvement in specific cellulase properties are directed evolution and knowledge-based library design based on multiple sequence alignments. In this chapter, we provide protocols for constructing and screening of improved thermostable cellulases. Modifications of these protocols may also be used for screening for other improved properties of cellulases such as pH tolerance, high salt, and more. Copyright © 2012 Elsevier Inc. All rights reserved.

A PCV2 vaccine based on genotype 2b is more effective than a 2a-based vaccine to protect against PCV2b or combined PCV2a/2b viremia in pigs with concurrent PCV2, PRRSV and PPV infection.

Science.gov (United States)

Opriessnig, Tanja; O'Neill, Kevin; Gerber, Priscilla F; de Castro, Alessandra M M G; Gimenéz-Lirola, Luis G; Beach, Nathan M; Zhou, Lei; Meng, Xiang-Jin; Wang, Chong; Halbur, Patrick G

2013-01-07

The predominant genotype of porcine circovirus (PCV) in the pig population today is PCV2b yet PCV2a-based commercial vaccines are considered effective in protecting against porcine circovirus associated disease. The objective of this study was to compare the ability of PCV2a- and PCV2b-based vaccines to control PCV2b viremia in a challenge model that mimics the U.S. field situation. Sixty-three pigs were randomly assigned to one of eight groups. Sixteen pigs were vaccinated with an experimental live-attenuated chimeric PCV1-2a vaccine based on genotype 2a and another 16 pigs with a chimeric PCV1-2b vaccine based on genotype 2b. Challenge was done 28 days post vaccination (dpv) using PCV2b (or a combination of PCV2a and PCV2b), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine parvovirus (PPV) to mimic what commonly occurs in the field. The experiment was terminated 21 days post challenge (dpc) or 49dpv. Pigs vaccinated with the chimeric PCV1-2b vaccine had significantly higher levels of PCV1-2b viremia and shedding of the PCV1-2b vaccine virus in feces and nasal secretions but also a more robust humoral immune response as evidenced by significantly higher ELISA S/P ratios compared to the PCV1-2a vaccination. Regardless of challenge, the PCV1-2b vaccination significantly reduced the prevalence and amount of PCV2 viremia compared to the PCV1-2a vaccination. Interestingly, in the non-vaccinated pigs concurrent PCV2a infection resulted in clinical disease and increased macroscopic lung lesions compared to pigs challenged with PCV2b alone, further supporting the idea that concurrent PCV2a/PCV2b infection is necessary for optimal PCV2 replication. Copyright © 2012 Elsevier Ltd. All rights reserved.

Engineering increased thermostability in the GH-10 endo-1,4-ß-xylanase from Thermoascus aurantiacus CBMAI 756

Science.gov (United States)

The GH10 endo-xylanase from Thermoascus aurantiacus CBMAI 756 (XynA) is industrially attractive due to its considerable thermostability and high specific activity. Considering the possibility of a further improvement in thermostability, eleven mutants were created in the present study via site-direc...

Comparative protection of two different commercial vaccines against Yersinia ruckeri serotype O1 and biotype 2 in rainbow trout (Oncorhynchus mykiss)

DEFF Research Database (Denmark)

Deshmukh, S.; Raida, M. K.; Dalsgaard, Inger

2012-01-01

Differentially extended specific protection by two commercial vaccines against Yersinia ruckeri serotype O1 biotype 2 was studied following 30s immersion exposure. Rainbow trout were challenged intra-peritoneally (i.p.) with Y. ruckeri serotype O1, biotype 2 (≈106 to 107CFU/fish) at 4, 6 and 8...... months after vaccination with vaccines containing either biotype 1 (AquaVac® ERM) or both biotypes 1 and 2 (AquaVac® RELERA™). The specific pattern of vaccine-mediated protection was evaluated by relative percentage survival (RPS) analysis at 4 and 6 months post-vaccination and by obtaining gross...

Improved thermostability and enzyme activity of a recombinant phyA mutant phytase from Aspergillus niger N25 by directed evolution and site-directed mutagenesis.

Science.gov (United States)

Tang, Zizhong; Jin, Weiqiong; Sun, Rong; Liao, Yan; Zhen, Tianrun; Chen, Hui; Wu, Qi; Gou, Lin; Li, Chenlei

2018-01-01

We previously constructed three recombinant phyA mutant strains (PP-NP m -8, PP-NP ep -6A and I44E/T252R-PhyA), showing improved catalytic efficiency or thermostability of Aspergillus niger N25 phytase, by error-prone PCR or site-directed mutagenesis. In this study, directed evolution and site-directed mutagenesis were further applied to improve the modified phytase properties. After one-round error-prone PCR for phytase gene of PP-NP ep -6A, a single transformant, T195L/Q368E/F376Y, was obtained with the significant improvements in catalytic efficiency and thermostability. The phytase gene of T195L/Q368E/F376Y, combined with the previous mutant phytase genes of PP-NP ep -6A, PP-NP m -8 and I44E/T252R-PhyA, was then sequentially modified by DNA shuffling. Three genetically engineered strains with desirable properties were then obtained, namedQ172R, Q172R/K432R andQ368E/K432R. Among them, Q172R/K432R showed the highest thermostability with the longest half-life and the greatest remaining phytase activity after heat treatment, while Q368E/K432R showed the highest catalytic activity. Five substitutions (Q172R, T195L, Q368E, F376Y, K432R) identified from random mutagenesis were added sequentially to the phytase gene of PP-NP ep -6A to investigate how the mutant sites influence the properties of phytase. Characterization and structural analysis demonstrated that these mutations could produce cumulative or synergistic improvements in thermostability or catalytic efficiency of phytase. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression and characterization of thermostable glycogen branching enzyme from Geobacillus mahadia Geo-05

Directory of Open Access Journals (Sweden)

Nur Syazwani Mohtar

2016-12-01

Full Text Available The glycogen branching enzyme (EC 2.4.1.18, which catalyses the formation of α-1,6-glycosidic branch points in glycogen structure, is often used to enhance the nutritional value and quality of food and beverages. In order to be applicable in industries, enzymes that are stable and active at high temperature are much desired. Using genome mining, the nucleotide sequence of the branching enzyme gene (glgB was extracted from the Geobacillus mahadia Geo-05 genome sequence provided by the Malaysia Genome Institute. The size of the gene is 2013 bp, and the theoretical molecular weight of the protein is 78.43 kDa. The gene sequence was then used to predict the thermostability, function and the three dimensional structure of the enzyme. The gene was cloned and overexpressed in E. coli to verify the predicted result experimentally. The purified enzyme was used to study the effect of temperature and pH on enzyme activity and stability, and the inhibitory effect by metal ion on enzyme activity. This thermostable glycogen branching enzyme was found to be most active at 55 °C, and the half-life at 60 °C and 70 °C was 24 h and 5 h, respectively. From this research, a thermostable glycogen branching enzyme was successfully isolated from Geobacillus mahadia Geo-05 by genome mining together with molecular biology technique.

A hyper-thermostable α-amylase from Pyrococcus furiosus accumulates in Nicotiana tabacum as functional aggregates.

Science.gov (United States)

Zhu, Hong; Reynolds, L Bruce; Menassa, Rima

2017-06-19

Alpha amylase hydrolyzes α-bonds of polysaccharides such as starch and produces malto-oligosaccharides. Its starch saccharification applications make it an essential enzyme in the textile, food and brewing industries. Commercially available α-amylase is mostly produced from Bacillus or Aspergillus. A hyper-thermostable and Ca 2++ independent α-amylase from Pyrococcus furiosus (PFA) expressed in E.coli forms insoluble inclusion bodies and thus is not feasible for industrial applications. We expressed PFA in Nicotiana tabacum and found that plant-produced PFA forms functional aggregates with an accumulation level up to 3.4 g/kg FW (fresh weight) in field conditions. The aggregates are functional without requiring refolding and therefore have potential to be applied as homogenized plant tissue without extraction or purification. PFA can also be extracted from plant tissue upon dissolution in a mild reducing buffer containing SDS. Like the enzyme produced in P. furiosus and in E. coli, plant produced PFA preserves hyper-thermophilicity and hyper-thermostability and has a long shelf life when stored in lyophilized leaf tissue. With tobacco's large biomass and high yield, hyper-thermostable α-amylase was produced at a scale of 42 kg per hectare. Tobacco may be a suitable bioreactor for industrial production of active hyperthermostable alpha amylase.

Vaccination of carp against SVCV with an oral DNA vaccine or an insect cells-based subunit vaccine.

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Embregts, C W E; Rigaudeau, D; Tacchi, L; Pijlman, G P; Kampers, L; Veselý, T; Pokorová, D; Boudinot, P; Wiegertjes, G F; Forlenza, M

2018-03-19

We recently reported on a successful vaccine for carp against SVCV based on the intramuscular injection of a DNA plasmid encoding the SVCV glycoprotein (SVCV-G). This shows that the intramuscular (i.m.) route of vaccination is suitable to trigger protective responses against SVCV, and that the SVCV G-protein is a suitable vaccine antigen. Yet, despite the general success of DNA vaccines, especially against fish rhabdoviruses, their practical implementation still faces legislative as well as consumer's acceptance concerns. Furthermore, the i.m. route of plasmid administration is not easily combined with most of the current vaccination regimes largely based on intraperitoneal or immersion vaccination. For this reason, in the current study we evaluated possible alternatives to a DNA-based i.m. injectable vaccine using the SVCV-G protein as the vaccine antigen. To this end, we tested two parallel approaches: the first based on the optimization of an alginate encapsulation method for oral delivery of DNA and protein antigens; the second based on the baculovirus recombinant expression of transmembrane SVCV-G protein in insect cells, administered as whole-cell subunit vaccine through the oral and injection route. In addition, in the case of the oral DNA vaccine, we also investigated the potential benefits of the mucosal adjuvants Escherichia coli lymphotoxin subunit B (LTB). Despite the use of various vaccine types, doses, regimes, and administration routes, no protection was observed, contrary to the full protection obtained with our reference i.m. DNA vaccine. The limited protection observed under the various conditions used in this study, the nature of the host, of the pathogen, the type of vaccine and encapsulation method, will therefore be discussed in details to provide an outlook for future vaccination strategies against SVCV. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Enhanced Thermostability of Arabidopsis Rubisco activase improves photosynthesis and growth rates under moderate heat stress.

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Kurek, Itzhak; Chang, Thom Kai; Bertain, Sean M; Madrigal, Alfredo; Liu, Lu; Lassner, Michael W; Zhu, Genhai

2007-10-01

Plant photosynthesis declines when the temperature exceeds its optimum range. Recent evidence indicates that the reduction in photosynthesis is linked to ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) deactivation due to the inhibition of Rubisco activase (RCA) under moderately elevated temperatures. To test the hypothesis that thermostable RCA can improve photosynthesis under elevated temperatures, we used gene shuffling technology to generate several Arabidopsis thaliana RCA1 (short isoform) variants exhibiting improved thermostability. Wild-type RCA1 and selected thermostable RCA1 variants were introduced into an Arabidopsis RCA deletion (Deltarca) line. In a long-term growth test at either constant 26 degrees C or daily 4-h 30 degrees C exposure, the transgenic lines with the thermostable RCA1 variants exhibited higher photosynthetic rates, improved development patterns, higher biomass, and increased seed yields compared with the lines expressing wild-type RCA1 and a slight improvement compared with untransformed Arabidopsis plants. These results provide clear evidence that RCA is a major limiting factor in plant photosynthesis under moderately elevated temperatures and a potential target for genetic manipulation to improve crop plants productivity under heat stress conditions.

Phase I clinical study of anti-apoptosis protein, survivin-derived peptide vaccine therapy for patients with advanced or recurrent colorectal cancer

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Minamida Hidetoshi

2004-06-01

Full Text Available Abstract Survivin is a member of the inhibitor of apoptosis protein (IAP family containing a single baculovirus IAP repeat domain. It is expressed during fetal development but becomes undetectable in terminally differentiated normal adult tissues. We previously reported that survivin and its splicing variant survivin-2B was expressed abundantly in various types of tumor tissues as well as tumor cell lines and was suitable as a target antigen for active-specific anti-cancer immunization. Subsequently, we identified an HLA-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL recognized by CD8+ cytotoxic T lymphocytes (CTLs. We, therefore, started a phase I clinical study assessing the efficacy of survivin-2B peptide vaccination in patients with advanced or recurrent colorectal cancer expressing survivin. Vaccinations with survivin-2B peptide were given subcutaneously six times at 14-day intervals. Of 15 patients who finished receiving the vaccination schedule, three suffered slight toxicities, including anemia (grade 2, general malaise (grade 1, and fever (grade 1. No severe adverse events were observed in any patient. In 6 patients, tumor marker levels (CEA and CA19-9 decreased transiently during the period of vaccination. Slight reduction of the tumor volume was observed in one patient, which was considered a minor responder. No changes were noted in three patients while the remaining eleven patients experienced tumor progression. Analysis of peripheral blood lymphocytes of one patient using HLA-A24/peptide tetramers revealed an increase in peptide-specific CTL frequency from 0.09% to 0.35% of CD8+ T cells after 4 vaccinations. This phase I clinical study indicates that survivin-2B peptide-based vaccination is safe and should be further considered for potential immune and clinical efficacy in HLA-A24-expression patients with colorectal cancer.

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

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Eun-Do Kim

Full Text Available 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.

Immunogenicity of an HPV-16 L2 DNA vaccine

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Hitzeroth, Inga I.; Passmore, Jo-Ann S.; Shephard, Enid; Stewart, Debbie; Müller, Martin; Williamson, Anna-Lise; Rybicki, Edward P.; Kast, W. Martin

2009-01-01

The ability to elicit cross-neutralizing antibodies makes human papillomavirus (HPV) L2 capsid protein a possible HPV vaccine. We examined and compared the humoral response of mice immunised with a HPV-16 L2 DNA vaccine or with HPV-16 L2 protein. The L2 DNA vaccine elicited a non-neutralising antibody response unlike the L2 protein. L2 DNA vaccination suppressed the growth of L2-expressing C3 tumor cells, which is a T cell mediated effect, demonstrating that the lack of non-neutralizing antibody induction by L2 DNA was not caused by lack of T cell immunogenicity of the construct. PMID:19559114

Expression of Acidothermus cellulolyticus thermostable cellulases in tobacco and rice plants

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Xiran Jiang

2017-01-01

Full Text Available The production of cellulases in plants is an economical method for the conversion of lignocellulosic biomass into fuels. Herein we report the expressions of two thermostable Acidothermus cellulolyticus cellulases, endo-1,4-β-D-glucanase (E1 and exoglucanase (Gux1, in tobacco and rice. To evaluate the expression of these recombinant cellulases, we expressed the full-length E1, the catalytic domains of E1 (E1cd and Gux1 (Gux1cd, as well as an E1–Gux1cd fusion enzyme in various subcellular compartments. In the case of tobacco, transgenic plants that expressed apoplast-localized E1 showed the highest level of activity, about three times higher than those that expressed the cytosolic E1. In the case of rice, the level of cellulase-specific activity in the transgenic plants ranged from 11 to 20 nmol 4-methylumbelliferone min−1 mg−1 total soluble protein. The recombinant cellulases exhibited good thermostability below 70 °C. Furthermore, transgenic rice leaves that were stored at room temperature for a month lost about 20% of the initial cellulase activity. Taken together, the results suggested that heterologous expression of thermostable cellulases in plants may be a viable option for biomass conversion.

HER-2 Pulsed Dendritic Cell Vaccine Can Eliminate HER-2 Expression and Impact DCIS

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Sharma, Anupama; Koldovsky, Ursula; Xu, Shuwen; Mick, Rosemarie; Roses, Robert; Fitzpatrick, Elizabeth; Weinstein, Susan; Nisenbaum, Harvey; Levine, Bruce L; Fox, Kevin; Zhang, Paul; Koski, Gary; Czerniecki, Brian J

2011-01-01

Background HER-2/neu over-expression plays a critical role in breast cancer development and its expression in ductal carcinoma in situ (DCIS) is associated with development of invasive breast cancer. A vaccine targeting HER-2/neu expression in DCIS may initiate immunity against invasive cancer. Methods A HER-2/neu dendritic cell (DC) vaccine was administered to 27 patients with HER-2/neu over-expressing DCIS. The HER-2/neu vaccine was administered prior to surgical resection and pre- and post-vaccination analysis was conducted to assess clinical results. Results At surgery, 5 of 27 (18.5%) vaccinated subjects had no evidence of remaining disease, while among 22 subjects with residual DCIS, HER-2/neu expression was eradicated in 11 (50%). When comparing ERneg with ERpos DCIS lesions, vaccination was more effective in hormone-independent DCIS. Following vaccination, no residual DCIS was found in 40% of ERneg subjects compared to 5.9% in ERpos subject. Sustained HER-2/neu expression was found in 10% of ERneg subjects compared to 47.1% in ERpos subjects (p=0.04). Post-vaccination phenotypes were significantly different between ERpos and ERneg subjects (p=0.01), with 7 of 16 (43.8%) initially presenting with ERpos HER-2/neupos Luminal B phenotype finishing with the ERpos HER-2/neuneg Luminal A phenotype, and 3 of 6 (50%) with the ERneg HER-2/neupos phenotype changing to the ERneg HER-2/neuneg phenotype. Conclusions Results suggest vaccination against HER-2/neu is safe, well-tolerated and induces decline and or eradication of HER-2/neu expression. These findings warrant further exploration of HER-2/neu vaccination in estrogen-independent breast cancer and highlight the need to target additional tumor associated antigens and pathways. PMID:22252842

Production of extremely alkaliphilic, halotolerent, detergent, and thermostable mannanase by the free and immobilized cells of Bacillus halodurans PPKS-2. Purification and characterization.

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Vijayalaxmi, S; Prakash, P; Jayalakshmi, S K; Mulimani, V H; Sreeramulu, K

2013-09-01

The alkaliphilic Bacillus halodurans strain PPKS-2 was shown to produce extracellular extreme alkaliphilic, halotolerent, detergent, and thermostable mannanase activity. The cultural conditions for the maximum enzyme production were optimized with respect to pH, temperature, NaCl, and inexpensive agro wastes as substrates. Mannanase production was enhanced more than 4-fold in the presence of 1 % defatted copra meal and 0.5 % peptone or feather hydrolysate at pH 11 and 40 °C. Mannanase was purified to 10.3-fold with 34.6 % yield by ion exchange and gel filtration chromatography methods. Its molecular mass was estimated to be 22 kDa by SDS-PAGE. The mannanase had maximal activity at pH 11 and 70 °C. This enzyme was active over a broad range of NaCl (0-16 %) and thermostable retaining 100 % of the original activity at 70 °C for 3 h. Immobilization of whole cells proved to be effective for continuous production of mannanase. Since the strain PPKS-2 grows on cheaper agro wastes such as defatted copra meal, corn husk, jowar bagasse, and wheat bran, these can be exploited for mannanase production on an industrial scale.

Effect of a phase I Coxiella burnetii inactivated vaccine on body temperature and milk yield in dairy cows.

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Schulze, L S-Ch; Borchardt, S; Ouellet, V; Heuwieser, W

2016-01-01

Q fever is a zoonotic disease caused by Coxiella burnetii. The pathogen is prevalent in ruminants (goats, sheep, cows), which are the main sources of human infection. In the cattle industry around the world, animal (15 to 20%) and herd (38 to 72%) level prevalences of C. burnetii are high. Vaccination of ruminants against Q fever is considered important to prevent spreading of the disease and risk of infection in humans. However, published information on side effects of the Q fever vaccination under field conditions is limited for cows. The objective of this study was to investigate the effect of the phase I C. burnetii inactivated vaccine Coxevac on body temperature and milk yield in dairy cows. In 2 experiments, a total of 508 cows were randomly divided into 2 groups to determine the effect of first vaccination on body temperature and milk yield. The C. burnetii serostatus of all cows was tested before vaccination with an indirect ELISA. The first experiment took place in the teaching and research barn of the Clinic of Animal Reproduction at the Freie Universität Berlin. Temperature was measured vaginally in 10 cows in a crossover design. The second experiment was conducted on a commercial dairy farm. Milk yield of 498 cows was measured 1 wk before and 1 wk after vaccination. In a subset of 41 cows, temperature was measured rectally. In both experiments, body temperature increased significantly after vaccination (1.0 ± 0.9°C and 0.7 ± 0.8°C). A significant difference was also found in body temperature between vaccinated and control cows. Thirty percent of the vaccinated animals in experiment 1 showed reversible swelling at the injection site as a reaction to the vaccination. The results indicate that vaccination against Q fever causes a transient increase of body temperature that peaks in the first 12 to 24h and declines after that. In experiment 2, vaccinated cows (26.8 ± 0.39 kg/d) produced significantly less milk than did control cows (28.2 ± 0.44 kg

Enhancement of thermo-stability and product tolerance of Pseudomonas putida nitrile hydratase by fusing with self-assembling peptide.

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Liu, Yi; Cui, Wenjing; Liu, Zhongmei; Cui, Youtian; Xia, Yuanyuan; Kobayashi, Michihiko; Zhou, Zhemin

2014-09-01

Self-assembling amphipathic peptides (SAPs) are the peptides that can spontaneously assemble into ordered nanostructures. It has been reported that the attachment of SAPs to the N- or C-terminus of an enzyme can benefit the thermo-stability of the enzyme. Here, we discovered that the thermo-stability and product tolerance of nitrile hydratase (NHase) were enhanced by fusing with two of the SAPs (EAK16 and ELK16). When the ELK16 was fused to the N-terminus of β-subunit, the resultant NHase (SAP-NHase-2) became an active inclusion body; EAK16 fused NHase in the N-terminus of β-subunit (SAP-NHase-1) and ELK16 fused NHase in the C-terminus of β-subunit (SAP-NHase-10) did not affect NHase solubility. Compared with the deactivation of the wild-type NHase after 30 min incubation at 50°C, SAP-NHase-1, SAP-NHase-2 and SAP-NHase-10 retained 45%, 30% and 50% activity; after treatment in the buffer containing 10% acrylamide, the wild-type retained 30% activity, while SAP-NHase-1, SAP-NHase-2 and SAP-NHase-10 retained 52%, 42% and 55% activity. These SAP-NHases with enhanced thermo-stability and product tolerance would be helpful for further industrial applications of the NHase. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Improvement in the thermostability of chitosanase from Bacillus ehimensis by introducing artificial disulfide bonds.

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Sheng, Jun; Ji, Xiaofeng; Zheng, Yuan; Wang, Zhipeng; Sun, Mi

2016-10-01

To determine the effects of artificial disulfide bridges on the thermostability and catalytic efficiency of chitosanase EAG1. Five artificial disulfide bridges were designed based on the structural information derived from the three-dimensional (3-D) model of chitosanase EAG1. Two beneficial mutants (G113C/D116C, A207C-L286C) were located in the flexible surface loop region, whereas the similar substitutions introduced in α-helices regions had a negligible effect. Mut5, the most active mutant, had a longer half-life at 50 °C (from 10.5 to 69.3 min) and a 200 % higher catalytic efficiency (K cat/K m) than that of the original EAG1. The contribution of disulfide bridges to enzyme thermostability is mainly dependent on its location within the polypeptide chain. Strategical placement of a disulfide bridge in flexible regions provides a rigid support and creation of a protected microenvironment, which is effective in improving enzyme's thermostability and catalytic efficiency.

Enhanced Thermostability of Arabidopsis Rubisco Activase Improves Photosynthesis and Growth Rates under Moderate Heat Stress[OA

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Kurek, Itzhak; Chang, Thom Kai; Bertain, Sean M.; Madrigal, Alfredo; Liu, Lu; Lassner, Michael W.; Zhu, Genhai

2007-01-01

Plant photosynthesis declines when the temperature exceeds its optimum range. Recent evidence indicates that the reduction in photosynthesis is linked to ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) deactivation due to the inhibition of Rubisco activase (RCA) under moderately elevated temperatures. To test the hypothesis that thermostable RCA can improve photosynthesis under elevated temperatures, we used gene shuffling technology to generate several Arabidopsis thaliana RCA1 (short isoform) variants exhibiting improved thermostability. Wild-type RCA1 and selected thermostable RCA1 variants were introduced into an Arabidopsis RCA deletion (Δrca) line. In a long-term growth test at either constant 26°C or daily 4-h 30°C exposure, the transgenic lines with the thermostable RCA1 variants exhibited higher photosynthetic rates, improved development patterns, higher biomass, and increased seed yields compared with the lines expressing wild-type RCA1 and a slight improvement compared with untransformed Arabidopsis plants. These results provide clear evidence that RCA is a major limiting factor in plant photosynthesis under moderately elevated temperatures and a potential target for genetic manipulation to improve crop plants productivity under heat stress conditions. PMID:17933901

Modern Vaccines/Adjuvants Formulation—Session 2 (Plenary II)

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Collin, Nicolas

2013-01-01

On the 15–17th May 2013, the Fourth International Conference on Modern Vaccines/Adjuvants Formulation was organized in Lausanne, Switzerland, and gathered stakeholders from academics and from the industry to discuss several challenges, advances and promises in the field of vaccine adjuvants. Plenary session 2 of the meeting was composed of four different presentations covering: (1) the recent set-up of an adjuvant technology transfer and training platform in Switzerland, (2) the proposition to revisit existing paradigms of modern vaccinology, (3) the properties of polyethyleneimine as potential new vaccine adjuvant, and (4) the progresses in the design of HIV vaccine candidates able to induce broadly neutralizing antibodies. PMID:23966098

Prime-boost BCG vaccination with DNA vaccines based in β-defensin-2 and mycobacterial antigens ESAT6 or Ag85B improve protection in a tuberculosis experimental model.

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Cervantes-Villagrana, Alberto R; Hernández-Pando, Rogelio; Biragyn, Arya; Castañeda-Delgado, Julio; Bodogai, Monica; Martínez-Fierro, Margarita; Sada, Eduardo; Trujillo, Valentin; Enciso-Moreno, Antonio; Rivas-Santiago, Bruno

2013-01-11

The World Health Organization (WHO) has estimated that there are about 8 million new cases annually of active Tuberculosis (TB). Despite its irregular effectiveness (0-89%), the Bacillus Calmette-Guérin) BCG is the only vaccine available worldwide for prevention of TB; thus, the design is important of novel and more efficient vaccination strategies. Considering that β-defensin-2 is an antimicrobial peptide that induces dendritic cell maturation through the TLR-4 receptor and that both ESAT-6 and Ag85B are immunodominant mycobacterial antigens and efficient activators of the protective immune response, we constructed two DNA vaccines by the fusion of the gene encoding β-defensin-2 and antigens ESAT6 (pDE) and 85B (pDA). After confirming efficient local antigen expression that induced high and stable Interferon gamma (IFN-γ) production in intramuscular (i.m.) vaccinated Balb/c mice, groups of mice were vaccinated with DNA vaccines in a prime-boost regimen with BCG and with BCG alone, and 2 months later were challenged with the mild virulence reference strain H37Rv and the highly virulent clinical isolate LAM 5186. The level of protection was evaluated by survival, lung bacilli burdens, and extension of tissue damage (pneumonia). Vaccination with both DNA vaccines showed similar protection to that of BCG. After the challenge with the highly virulent Mycobacterium tuberculosis strain, animals that were prime-boosted with BCG and then boosted with both DNA vaccines showed significant higher survival and less tissue damage than mice vaccinated only with BCG. These results suggest that improvement of BCG vaccination, such as the prime-boost DNA vaccine, represents a more efficient vaccination scheme against TB. Copyright © 2012 Elsevier Ltd. All rights reserved.

A comparative molecular dynamics study on thermostability of human and chicken prion proteins

International Nuclear Information System (INIS)

Ji, Hong-Fang; Zhang, Hong-Yu

2007-01-01

To compare the thermostabilities of human and chicken normal cellular prion proteins (HuPrP C and CkPrP C ), molecular dynamics (MD) simulations were performed for both proteins at an ensemble level (10 parallel simulations at 400 K and 5 parallel simulations at 300 K as a control). It is found that the thermostability of HuPrP C is comparable with that of CkPrP C , which implicates that the non-occurrence of prion diseases in non-mammals cannot be completely attributed to the thermodynamic properties of non-mammalian PrP C

Hot or not? Discovery and characterization of a thermostable alditol oxidase from Acidothermus cellulolyticus 11B

NARCIS (Netherlands)

Winter, Remko T.; Heuts, Dominic P. H. M.; Rijpkema, Egon M. A.; van Bloois, Edwin; Wijma, Hein J.; Fraaije, Marco W.

We describe the discovery, isolation and characterization of a highly thermostable alditol oxidase from Acidothermus cellulolyticus 11B. This protein was identified by searching the genomes of known thermophiles for enzymes homologous to Streptomyces coelicolor A3(2) alditol oxidase (AldO). A gene

Vaccination and Infection as Causative Factors in Japanese Patients With Rasmussen Syndrome: Molecular Mimicry and HLA Class I

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Yukitoshi Takahashi

2006-01-01

Full Text Available Rasmussen syndrome is an intractable epilepsy with a putative causal relation with cellular and humoral autoimmunity. Almost half of the patients have some preceding causative factors, with infections found in 38.2%, vaccinations in 5.9% and head trauma in 8.9% of Japanese patients. In a patient with seizure onset after influenza A infections, cross-reaction of the patient's lymphocytes with GluRε2 and influenza vaccine components was demonstrated by lymphocyte stimulation test. Database analyses revealed that influenza A virus hemagglutinin and GluRε2 molecules contain peptides with the patient's HLA class I binding motif (HLA ࢤ A*0201. The relative risks of HLA class I genotypes for Rasmussen syndrome are 6.1 (A*2402, 6.4 (A*0201, 6.3 (A*2601 and 11.4 (B*4601. The relative risks of HLA class I-A and B haplotypes are infinity (A*2601+B*5401, 21.1 (A*2402+B*1501, 13.3 (A*2402+B*4801 and 5.1 (A*2402+B*5201. Some alleles and haplotypes of HLA class I may be the risk factors in Japanese patients. Cross-reactivity of cytotoxic T lymphocytes may contribute to the processes leading from infection to the involvement of CNS.

Protective immunity conferred by porcine circovirus 2 ORF2-based DNA vaccine in mice.

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Sylla, Seydou; Cong, Yan-Long; Sun, Yi-Xue; Yang, Gui-Lian; Ding, Xue-Mei; Yang, Zhan-Qing; Zhou, Yu-Long; Yang, Minnan; Wang, Chun-Feng; Ding, Zhuang

2014-07-01

Post-weaning multisystemic wasting syndrome (PMWS) associated with porcine circovirus type 2 (PCV2) has caused the swine industry significant health challenges and economic damage. Although inactivated and subunit vaccines against PMWS have been used widely, so far no DNA vaccine is available. In this study, with the aim of exploring a new route for developing a vaccine against PCV2, the immunogenicity of a DNA vaccine was evaluated in mice. The pEGFP-N1 vector was used to construct a PCV2 Cap gene recombinant vaccine. To assess the immunogenicity of pEGFP-Cap, 80 BALB/c mice were immunized three times at 2 weekly intervals with pEGFP-Cap, LG-strain vaccine, pEGFP-N1 vector or PBS and then challenged with PCV2. IgG and cytokines were assessed by indirect ELISA and ELISA, respectively. Specimens stained with hematoxylin and eosin (HE) and immunohistochemistry (IHC) techniques were examined histopathologically. It was found that vaccination of the mice with the pEGFP-Cap induced solid protection against PCV2 infection through induction of highly specific serum IgG antibodies and cytokines (IFN-γ and IL-10), and a small PCV2 viral load. The mice treated with the pEGFP-Cap and LG-strain developed no histopathologically detectable lesions (HE stain) and IHC techniques revealed only a few positive cells. Thus, this study demonstrated that recombinant pEGFP-Cap substantially alleviates PCV2 infection in mice and provides evidence that a DNA vaccine could be an alternative to PCV2 vaccines against PMWS. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

Safety and long-term immunological effects of CryJ2-LAMP plasmid vaccine in Japanese red cedar atopic subjects: A phase I study.

Science.gov (United States)

Su, Yan; Romeu-Bonilla, Eliezer; Anagnostou, Athanasia; Fitz-Patrick, David; Hearl, William; Heiland, Teri

2017-12-02

Japanese Red Cedar (JRC) pollen induced allergy affects one third of Japanese and the development of effective therapies remains an unachieved challenge. We designed a DNA vaccine encoding CryJ2 allergen from the JRC pollen and Lysosomal Associated Membrane Protein 1 (LAMP-1) to treat JRC allergy. These Phase IA and IB trials assessed safety and immunological effects of the investigational CryJ2-LAMP DNA vaccine in both non-sensitive and sensitive Japanese expatriates living in Honolulu, Hawaii. In the Phase IA trial, 6 JRC non-sensitive subjects and 9 JRC and/or Mountain Cedar (MC) sensitive subjects were given 4 vaccine doses (each 4mg/1ml) intramuscularly (IM) at 14-day intervals. Nine JRC and/or MC sensitive subjects were given 4 doses (2 mg/0.5 ml) IM at 14-day intervals. The safety and functional biomarkers were followed for 132 d. Following this, 17 of 24 subjects were recruited into the IB trial and received one booster dose (2 mg/0.5 ml) IM approximately 300 d after the first vaccination dose to which they were randomized in the first phase of the trial. All safety endpoints were met and all subjects tolerated CryJ2-LAMP vaccinations well. At the end of the IA trial, 10 out of 12 JRC sensitive and 6 out of 11 MC sensitive subjects experienced skin test negative conversion, possibly related to the CryJ2-LAMP vaccinations. Collectively, these data suggested that the CryJ2-LAMP DNA vaccine is safe and may be immunologically effective in treating JRC induced allergy.

Thermostable cellulase from a thermomonospora gene

Science.gov (United States)

Wilson, D.B.; Walker, L.P.; Zhang, S.

1997-10-14

The invention relates to a gene isolated from Thermomonospora fusca, wherein the gene encodes a thermostable cellulase. Disclosed is the nucleotide sequence of the T. fusca gene; and nucleic acid molecules comprising the gene, or a fragment of the gene, that can be used to recombinantly express the cellulase or a catalytically active polypeptide thereof, respectively. The isolated and purified recombinant cellulase or catalytically active polypeptide may be used to hydrolyze substrate either by itself; or in combination with other cellulases, with the resultant combination having unexpected hydrolytic activity. 3 figs.

Canine parvovirus type 2 vaccine protects against virulent challenge with type 2c virus.

Science.gov (United States)

Spibey, N; Greenwood, N M; Sutton, D; Chalmers, W S K; Tarpey, I

2008-04-01

The ability of dogs vaccinated with a live attenuated CPV type 2 (Nobivac Intervet) vaccine to resist challenge with a current CPV2c isolate was investigated. Six SPF beagle dogs were given the minimum recommended course of vaccination, comprising a single inoculation of vaccine (Nobivac Lepto+Nobivac Pi) at 8-10 weeks of age followed 3 weeks later with a parvovirus vaccine in combination with distemper, adenovirus and parainfluenza virus (Nobivac DHPPi) and a repeat leptospirosis vaccine. Six control dogs were kept unvaccinated. All animals were challenged orally with a type 2c isolate of CPV and monitored for clinical signs, virus shedding, white blood cell fluctuations and serological responses. All vaccinated dogs were fully protected; showing no clinical signs nor shedding challenge virus in the faeces, in contrast to control animals, which displayed all the typical signs of infection with pathogenic CPV and shed challenge virus in the faeces.

FireProt: web server for automated design of thermostable proteins

Science.gov (United States)

Musil, Milos; Stourac, Jan; Brezovsky, Jan; Prokop, Zbynek; Zendulka, Jaroslav; Martinek, Tomas

2017-01-01

Abstract There is a continuous interest in increasing proteins stability to enhance their usability in numerous biomedical and biotechnological applications. A number of in silico tools for the prediction of the effect of mutations on protein stability have been developed recently. However, only single-point mutations with a small effect on protein stability are typically predicted with the existing tools and have to be followed by laborious protein expression, purification, and characterization. Here, we present FireProt, a web server for the automated design of multiple-point thermostable mutant proteins that combines structural and evolutionary information in its calculation core. FireProt utilizes sixteen tools and three protein engineering strategies for making reliable protein designs. The server is complemented with interactive, easy-to-use interface that allows users to directly analyze and optionally modify designed thermostable mutants. FireProt is freely available at http://loschmidt.chemi.muni.cz/fireprot. PMID:28449074

A novel inactivated gE/gI deleted pseudorabies virus (PRV) vaccine completely protects pigs from an emerged variant PRV challenge.

Science.gov (United States)

Gu, Zhenqing; Dong, Jing; Wang, Jichun; Hou, Chengcai; Sun, Haifeng; Yang, Wenping; Bai, Juan; Jiang, Ping

2015-01-02

A highly virulent and antigenic variant of pseudorabies virus (PRV) broke out in China at the end of 2011 and caused great economic loss in the pig industry. In this study, an infectious bacterial artificial chromosome (BAC) clone containing the full-length genome of the emerged variant PRV ZJ01 strain was generated. The BAC-derived viruses, vZJ01-GFPΔgE/gI (gE/gI deleted strain, and exhibiting green autofluorescence), vZJ01ΔgE/gI (gE/gI deleted strain), and vZJ01gE/gI-R (gE/gI revertant strain), showed similar in vitro growth to their parent strain. In pigs, inactivated vZJ01ΔgE/gI vaccine generated significantly high levels of neutralizing antibodies against ZJ01 compared with Bartha-K61 live vaccine (pvaccine group survived without exhibiting any clinical sings, but two of five animals exhibited central nervous signs in the Bartha-K61 group. Meanwhile, all the non-vaccinated control animals died at 7 days post-challenge. This indicates that the inactivated vZJ01ΔgE/gI vaccine is a promising vaccine candidate for controlling the variant strains of PRV now circulating in China. Copyright © 2014 Elsevier B.V. All rights reserved.

'THERMOST' for analysing thermo-structural behaviour of LWR fuel rods under PCI conditions

International Nuclear Information System (INIS)

Nuno, H.; Ogawa, S.; Kobayashi, H.

1983-01-01

As a method for evaluating fuel rod performance under power ramping or load following operations, the combined FROST/ THERMOST system has been developed and brought into practical use. FROST was presented at the IAEA Blackpool Meeting in 1978, and THERMOST is the subject of this paper. The major purpose of THERMOST is to analyse very detailed thermal and structural fuel behaviour in a rather localised part of the fuel rod whereas FROST deals with whole rod general performance. The code handles two-dimensional thermal and structural analyses simultaneously by using a finite element method, in axial section or in lateral section. It consists of a fundamental FEM system of generalised constitution, and a surrounding subroutine system which characterises fuel behaviour, such as temperature distribution, thermal expansion, elastoplasticity, creep, cracking, swelling, growth, etc. Thermal analysis is handled by heat conduction and heat transfer element (six kinds), and structural analysis by axisymmetric ring and lateral plane element (six kinds). Boundary problems such as contact, friction and cracking are treated by gap and crack elements. A sample calculation of PCI performance on a PWR fuel rod under ramping conditions is presented with some in-pile test data. (author)

Three-year serologic immunity against canine parvovirus type 2 and canine adenovirus type 2 in dogs vaccinated with a canine combination vaccine.

Science.gov (United States)

Larson, L J; Schultz, R D

2007-01-01

A group of client-owned dogs and a group of dogs at a commercial kennel were evaluated for duration of antibody responses against canine parvovirus type 2 (CPV-2) and canine adenovirus type 1 (CAV-1) after receiving a combination vaccine containing recombinant canarypox-vectored canine distemper virus (CDV) and modified-live CPV-2, CAV-2, and canine parainfluenza virus, with (C6) or without (C4) two serovars of Leptospira (Recombitek C4 or C6, Merial). Duration of antibody, which correlates with protective immunity, was found to be at least 36 months in both groups. Recombitek combination vaccines can confidently be given every 3 years with assurance of protection in immunocompetent dogs against CPV-2 and CAV-1 as well as CDV. This allows this combination vaccine, like other, similar modified- live virus combination products containing CDV, CAV-2, and CPV-2, to be administered in accordance with the recommendations of the American Animal Hospital Association Canine Vaccine Task Force.

Detection of FMD virus type specific IgG1, IgG2 and IgA antibodies in milk and serum of buffaloes vaccinated with oil adjuvanted polyvalent FMD vaccine

Directory of Open Access Journals (Sweden)

R. Sharma

2010-02-01

Full Text Available The present investigation was carried out on 15 randomly selected milch buffaloes divided into three groups on the basis of lactation at an organized farm, to study the foot and mouth disease virus type specific antibodies in milk and serum following FMD vaccination. Milk and serum samples collected before vaccination i.e. 0 day and on 7, 14, 28, 42 and 56 days post vaccination, were analyzed for the detection of FMD virus specific IgG1, IgG2 and IgA antibody response by indirect double antibody sandwich ELISA. Significant FMD virus type specific antibody titres (IgG1, IgG2 and IgA were detected in milk and serum of buffaloes on different days post vaccination, though the levels of antibodies were lower in milk as compared to serum. FMD virus type specific IgG1 was found to be the predominant subclass as compared to IgG2 and IgA both in milk and serum of vaccinated buffaloes. Milk and serum IgG1, IgG2 and IgA antibody titres were positively correlated with values of regression coefficient (R as 0.506, 0.434 and 0.396, respectively.

Adenovirus 2, Bordetella bronchiseptica, and Parainfluenza Molecular Diagnostic Assay Results in Puppies After vaccination with Modified Live Vaccines.

Science.gov (United States)

Ruch-Gallie, R; Moroff, S; Lappin, M R

2016-01-01

Canine adenovirus 2, parainfluenza, and Bordetella bronchiseptica cause respiratory disease in dogs, and each has a modified live intranasal vaccine available. Molecular diagnostic assays to amplify specific nucleic acids are available for each of these agents. If positive molecular diagnostic assay results are common after vaccination, the positive predictive value of the diagnostic assays for disease would be decreased. To determine the impact of administration of commercially available modified live topical adenovirus 2, B. bronchiseptica, and parainfluenza vaccine has on the results of a commercially available PCR panel. Eight puppies from a research breeding facility negative for these pathogens. Blinded prospective pilot study. Puppies were vaccinated with a single dose of modified live topical adenovirus 2, B. bronchiseptica, and parainfluenza and parenteral dose of adenovirus 2, canine distemper virus, and parvovirus. Nasal and pharyngeal swabs were collected on multiple days and submitted for PCR assay. Nucleic acids of all 3 organisms contained in the topical vaccine were detected from both samples multiple times through 28 days after vaccination with higher numbers of positive samples detected between days 3 and 10 after vaccination. Vaccine status should be considered when interpreting respiratory agent PCR results if modified live vaccines have been used. Development of quantitative PCR and wild-type sequencing are necessary to improve positive predictive value of these assays by distinguishing vaccinate from natural infection. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

Haematopoietic depletion in vaccine-induced neonatal pancytopenia depends on both the titre and specificity of alloantibody and levels of MHC I expression.

Science.gov (United States)

Bell, Charlotte R; MacHugh, Niall D; Connelley, Timothy K; Degnan, Kathryn; Morrison, W Ivan

2015-07-09

Bovine Neonatal Pancytopenia (BNP) is a disease of calves characterised by haematopoietic depletion, mediated by ingestion of alloantibodies in colostrum. It has been linked epidemiologically to vaccination of the dams of affected calves with a particular vaccine (Pregsure) containing a novel adjuvant. Evidence suggests that BNP-alloantibodies are directed against MHC I molecules, induced by contaminant bovine cellular material from Madin-Darby Bovine Kidney (MDBK) cells used in the vaccine's production. We aimed to investigate the specificity of BNP-alloantibody for bovine MHC I alleles, particularly those expressed by MDBK cells, and whether depletion of particular cell types is due to differential MHC I expression levels. A complement-mediated cytotoxicity assay was used to assess functional serum alloantibody titres in BNP-dams, Pregsure-vaccinated dams with healthy calves, cows vaccinated with an alternative product and unvaccinated controls. Alloantibody specificity was investigated using transfected mouse lines expressing the individual MHC I alleles identified from MDBK cells and MHC I-defined bovine leukocyte lines. All BNP-dams and 50% of Pregsure-vaccinated cows were shown to have MDBK-MHC I specific alloantibodies, which cross-reacted to varying degrees with other MHC I genotypes. MHC I expression levels on different blood cell types, assessed by flow cytometry, were found to correlate with levels of alloantibody-mediated damage in vitro and in vivo. Alloantibody-killed bone marrow cells were shown to express higher levels of MHC I than undamaged cells. The results provide evidence that MHC I-specific alloantibodies play a dominant role in the pathogenesis of BNP. Haematopoietic depletion was shown to be dependent on the titre and specificity of alloantibody produced by individual cows and the density of surface MHC I expression by different cell types. Collectively, the results support the hypothesis that MHC I molecules originating from MDBK cells

In Vitro Characterization of Thermostable CAM Rubisco Activase Reveals a Rubisco Interacting Surface Loop.

Science.gov (United States)

Shivhare, Devendra; Mueller-Cajar, Oliver

2017-07-01

To maintain metabolic flux through the Calvin-Benson-Bassham cycle in higher plants, dead-end inhibited complexes of Rubisco must constantly be engaged and remodeled by the molecular chaperone Rubisco activase (Rca). In C3 plants, the thermolability of Rca is responsible for the deactivation of Rubisco and reduction of photosynthesis at moderately elevated temperatures. We reasoned that crassulacean acid metabolism (CAM) plants must possess thermostable Rca to support Calvin-Benson-Bassham cycle flux during the day when stomata are closed. A comparative biochemical characterization of rice ( Oryza sativa ) and Agave tequilana Rca isoforms demonstrated that the CAM Rca isoforms are approximately10°C more thermostable than the C3 isoforms. Agave Rca also possessed a much higher in vitro biochemical activity, even at low assay temperatures. Mixtures of rice and agave Rca form functional hetero-oligomers in vitro, but only the rice isoforms denature at nonpermissive temperatures. The high thermostability and activity of agave Rca mapped to the N-terminal 244 residues. A Glu-217-Gln amino acid substitution was found to confer high Rca activity to rice Rca Further mutational analysis suggested that Glu-217 restricts the flexibility of the α4-β4 surface loop that interacts with Rubisco via Lys-216. CAM plants thus promise to be a source of highly functional, thermostable Rca candidates for thermal fortification of crop photosynthesis. Careful characterization of their properties will likely reveal further protein-protein interaction motifs to enrich our mechanistic model of Rca function. © 2017 American Society of Plant Biologists. All Rights Reserved.

Potential and utilization of thermophiles and thermostable enzymes in biorefining

Directory of Open Access Journals (Sweden)

Karlsson Eva

2007-03-01

Full Text Available Abstract In today's world, there is an increasing trend towards the use of renewable, cheap and readily available biomass in the production of a wide variety of fine and bulk chemicals in different biorefineries. Biorefineries utilize the activities of microbial cells and their enzymes to convert biomass into target products. Many of these processes require enzymes which are operationally stable at high temperature thus allowing e.g. easy mixing, better substrate solubility, high mass transfer rate, and lowered risk of contamination. Thermophiles have often been proposed as sources of industrially relevant thermostable enzymes. Here we discuss existing and potential applications of thermophiles and thermostable enzymes with focus on conversion of carbohydrate containing raw materials. Their importance in biorefineries is explained using examples of lignocellulose and starch conversions to desired products. Strategies that enhance thermostablity of enzymes both in vivo and in vitro are also assessed. Moreover, this review deals with efforts made on developing vectors for expressing recombinant enzymes in thermophilic hosts.

Herpes simplex virus-2 in the genital mucosa: insights into the mucosal host response and vaccine development.

Science.gov (United States)

Lee, Amanda J; Ashkar, Ali A

2012-02-01

Herpes simplex virus (HSV)-2 is the predominant cause of genital herpes and has been implicated in HIV infection and transmission. Thus far, vaccines developed against HSV-2 have been clinically ineffective in preventing infection. This review aims to summarize the innate and adaptive immune responses against HSV-2 and examines the current status of vaccine development. Both innate and adaptive immune responses are essential for an effective primary immune response and the generation of immunity. The innate response involves Toll-like receptors, natural killer cells, plasmacytoid dendritic cells, and type I, II, and III interferons. The adaptive response requires a balance between CD4+ and CD8+ T-cells for optimal viral clearance. T-regulatory cells may be involved, although their exact function has yet to be determined. Current vaccine development involves the use of HSV-2 peptides or attenuated/replication-defective HSV-2 to generate adaptive anti-HSV-2 immune responses, however the generation of innate responses may also be an important consideration. Although vaccine development has primarily focused on the adaptive response, arguments for innate involvement are emerging. A greater understanding of the innate and adaptive processes underlying the response to HSV-2 infection will provide the foundation for the development of an effective vaccine.

An E2-Substituted Chimeric Pestivirus With DIVA Vaccine Properties

DEFF Research Database (Denmark)

Rasmussen, Thomas Bruun; Uttenthal, Åse; Nielsen, Jens

An advantage of the use of chimeric pestiviruses as modified live vaccines against classical swine fever (CSF) resides in their capacity to be manipulated to achieve the characteristics desired for safe and efficacious DIVA vaccines. We have recently generated a new chimeric virus, Riems26_E2gif...... vaccinated pigs were protected. This new chimeric pestivirus represents a C-strain based DIVA vaccine candidate that can be differentiated based on CSFV E2 specific antibodies....

Clinical and immunological evaluation of anti-apoptosis protein, survivin-derived peptide vaccine in phase I clinical study for patients with advanced or recurrent breast cancer

Directory of Open Access Journals (Sweden)

Asanuma Hiroko

2008-05-01

Full Text Available Abstract Background We previously reported that survivin-2B, a splicing variant of survivin, was expressed in various types of tumors and that survivin-2B peptide might serve as a potent immunogenic cancer vaccine. The objective of this study was to examine the toxicity of and to clinically and immunologically evaluate survivin-2B peptide in a phase I clinical study for patients with advanced or recurrent breast cancer. Methods We set up two protocols. In the first protocol, 10 patients were vaccinated with escalating doses (0.1–1.0 mg of survivin-2B peptide alone 4 times every 2 weeks. In the second protocol, 4 patients were vaccinated with the peptide at a dose of 1.0 mg mixed with IFA 4 times every 2 weeks. Results In the first protocol, no adverse events were observed during or after vaccination. In the second protocol, two patients had induration at the injection site. One patient had general malaise (grade 1, and another had general malaise (grade 1 and fever (grade 1. Peptide vaccination was well tolerated in all patients. In the first protocol, tumor marker levels increased in 8 patients, slightly decreased in 1 patient and were within the normal range during this clinical trial in 1 patient. With regard to tumor size, two patients were considered to have stable disease (SD. Immunologically, in 3 of the 10 patients (30%, an increase of the peptide-specific CTL frequency was detected. In the second protocol, an increase of the peptide-specific CTL frequency was detected in all 4 patients (100%, although there were no significant beneficial clinical responses. ELISPOT assay showed peptide-specific IFN-γ responses in 2 patients in whom the peptide-specific CTL frequency in tetramer staining also was increased in both protocols. Conclusion This phase I clinical study revealed that survivin-2B peptide vaccination was well tolerated. The vaccination with survivin-2B peptide mixed with IFA increased the frequency of peptide-specific CTL more

PREPARATION AND PROPERTIES OF THREE NEUTRAL COPPER (I COMPLEXES BASED ON BIS[2-(DIPHENYLPHOSPHINOPHENYL]ETHER PHENYL] ETHER AND SUBSTITUTED IMIDAZOLE-2,9-DIMETHYL-1,10-PHENANTHROLINE

Directory of Open Access Journals (Sweden)

Xin Fang Liu

2017-10-01

Full Text Available With deprotonated 2-(4-nitro phenyl-, 2-(4-methyl phenyl-, and 2-(4-methoxy phenyl imidazoled-2,9-dimethyl-1,10-phenanthroline (NPIP, MPIP and MoPIP as nitrogen ligands, as well as bis[2-(diphenylphosphino phenyl]ether (DPEphos as phosphorus ligand, three Cu(I neutral complex has been synthesized and characterized by infrared spectroscopy (IR, elemental analysis and 1H NMR methods. TG-DTA shows that the complex begins to decompose when the temperature reached about 220 oC, indicating their high thermo-stability. The emission spectrum shows that the complexes exhibit yellow emission with a peak emission wavelength of 576, 585 and 596 nm under excitation of 287 nm in powder state.

Vaccination with EphA2-derived T cell-epitopes promotes immunity against both EphA2-expressing and EphA2-negative tumors

Science.gov (United States)

Hatano, Manabu; Kuwashima, Naruo; Tatsumi, Tomohide; Dusak, Jill E; Nishimura, Fumihiko; Reilly, Karlyne M; Storkus, Walter J; Okada, Hideho

2004-01-01

Background A novel tyrosine kinase receptor EphA2 is expressed at high levels in advanced and metastatic cancers. We examined whether vaccinations with synthetic mouse EphA2 (mEphA2)-derived peptides that serve as T cell epitopes could induce protective and therapeutic anti-tumor immunity. Methods C57BL/6 mice received subcutaneous (s.c.) vaccinations with bone marrow-derived dendritic cells (DCs) pulsed with synthetic peptides recognized by CD8+ (mEphA2671–679, mEphA2682–689) and CD4+ (mEphA230–44) T cells. Splenocytes (SPCs) were harvested from primed mice to assess the induction of cytotoxic T lymphocyte (CTL) responses against syngeneic glioma, sarcoma and melanoma cell lines. The ability of these vaccines to prevent or treat tumor (s.c. injected MCA205 sarcoma or B16 melanoma; i.v. injected B16-BL6) establishment/progression was then assessed. Results Immunization of C57BL/6 mice with mEphA2-derived peptides induced specific CTL responses in SPCs. Vaccination with mEPhA2 peptides, but not control ovalbumin (OVA) peptides, prevented the establishment or prevented the growth of EphA2+ or EphA2-negative syngeneic tumors in both s.c. and lung metastasis models. Conclusions These data indicate that mEphA2 can serve as an attractive target against which to direct anti-tumor immunity. The ability of mEphA2 vaccines to impact EphA2-negative tumors such as the B16 melanoma may suggest that such beneficial immunity may be directed against alternative EphA2+ target cells, such as the tumor-associated vascular endothelial cells. PMID:15563374

Vaccination with EphA2-derived T cell-epitopes promotes immunity against both EphA2-expressing and EphA2-negative tumors

Directory of Open Access Journals (Sweden)

Hatano Manabu

2004-11-01

Full Text Available Abstract Background A novel tyrosine kinase receptor EphA2 is expressed at high levels in advanced and metastatic cancers. We examined whether vaccinations with synthetic mouse EphA2 (mEphA2-derived peptides that serve as T cell epitopes could induce protective and therapeutic anti-tumor immunity. Methods C57BL/6 mice received subcutaneous (s.c. vaccinations with bone marrow-derived dendritic cells (DCs pulsed with synthetic peptides recognized by CD8+ (mEphA2671–679, mEphA2682–689 and CD4+ (mEphA230–44 T cells. Splenocytes (SPCs were harvested from primed mice to assess the induction of cytotoxic T lymphocyte (CTL responses against syngeneic glioma, sarcoma and melanoma cell lines. The ability of these vaccines to prevent or treat tumor (s.c. injected MCA205 sarcoma or B16 melanoma; i.v. injected B16-BL6 establishment/progression was then assessed. Results Immunization of C57BL/6 mice with mEphA2-derived peptides induced specific CTL responses in SPCs. Vaccination with mEPhA2 peptides, but not control ovalbumin (OVA peptides, prevented the establishment or prevented the growth of EphA2+ or EphA2-negative syngeneic tumors in both s.c. and lung metastasis models. Conclusions These data indicate that mEphA2 can serve as an attractive target against which to direct anti-tumor immunity. The ability of mEphA2 vaccines to impact EphA2-negative tumors such as the B16 melanoma may suggest that such beneficial immunity may be directed against alternative EphA2+ target cells, such as the tumor-associated vascular endothelial cells.

Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties

Directory of Open Access Journals (Sweden)

Koh Eunhee

2007-07-01

Full Text Available Abstract Background EstE1 is a hyperthermophilic esterase belonging to the hormone-sensitive lipase family and was originally isolated by functional screening of a metagenomic library constructed from a thermal environmental sample. Dimers and oligomers may have been evolutionally selected in thermophiles because intersubunit interactions can confer thermostability on the proteins. The molecular mechanisms of thermostabilization of this extremely thermostable esterase are not well understood due to the lack of structural information. Results Here we report for the first time the 2.1-Å resolution crystal structure of EstE1. The three-dimensional structure of EstE1 exhibits a classic α/β hydrolase fold with a central parallel-stranded beta sheet surrounded by alpha helices on both sides. The residues Ser154, Asp251, and His281 form the catalytic triad motif commonly found in other α/β hydrolases. EstE1 exists as a dimer that is formed by hydrophobic interactions and salt bridges. Circular dichroism spectroscopy and heat inactivation kinetic analysis of EstE1 mutants, which were generated by structure-based site-directed mutagenesis of amino acid residues participating in EstE1 dimerization, revealed that hydrophobic interactions through Val274 and Phe276 on the β8 strand of each monomer play a major role in the dimerization of EstE1. In contrast, the intermolecular salt bridges contribute less significantly to the dimerization and thermostability of EstE1. Conclusion Our results suggest that intermolecular hydrophobic interactions are essential for the hyperthermostability of EstE1. The molecular mechanism that allows EstE1 to endure high temperature will provide guideline for rational design of a thermostable esterase/lipase using the lipolytic enzymes showing structural similarity to EstE1.

Resolution Mechanism and Characterization of an Ammonium Chloride-Tolerant, High-Thermostable, and Salt-Tolerant Phenylalanine Dehydrogenase from Bacillus halodurans.

Science.gov (United States)

Jiang, Wei; Wang, Ya-Li; Fang, Bai-Shan

2018-05-09

As phenylalanine dehydrogenase (PheDH) plays an important role in the synthesis of chiral drug intermediates and detection of phenylketonuria, it is significant to obtain a PheDH with specific and high activity. Here, a PheDH gene, pdh, encoding a novel BhPheDH with 61.0% similarity to the known PheDH from Microbacterium sp., was obtained. The BhPheDH showed optimal activity at 60 °C and pH 7.0, and it showed better stability in hot environment (40-70 °C) than the PheDH from Nocardia sp. And its activity and thermostability could be significantly increased by sodium salt. After incubation for 2 h in 3 M NaCl at 60 °C, the residual activity of the BhPheDH was found to be 1.8-fold higher than that of the control group (without NaCl). The BhPheDH could tolerate high concentration of ammonium chloride and its activity could be also enhanced by the high concentration of ammonium salts. These characteristics indicate that the BhPheDH possesses better thermostability, ammonium chloride tolerance, halophilic mechanism, and high salt activation. The mechanism of thermostability and high salt tolerance of the BhPheDH was analyzed by molecular dynamics simulation. These results provide useful information about the enzyme with high-temperature activity, thermostability, halophilic mechanism, tolerance to high concentration of ammonium chloride, higher salt activation and enantio-selectivity, and the application of molecular dynamics simulation in analyzing the mechanism of these distinctive characteristics.

Induction of broad immunity by thermostabilised vaccines incorporated in dissolvable microneedles using novel fabrication methods.

Science.gov (United States)

Vrdoljak, Anto; Allen, Evin A; Ferrara, Francesca; Temperton, Nigel J; Crean, Abina M; Moore, Anne C

2016-03-10

Dissolvable microneedle (DMN) patches for immunization have multiple benefits, including vaccine stability and ease-of-use. However, conventional DMN fabrication methods have several drawbacks. Here we describe a novel, microfluidic, drop dispensing-based dissolvable microneedle production method that overcomes these issues. Uniquely, heterogeneous arrays, consisting of microneedles of diverse composition, can be easily produced on the same patch. Robustness of the process was demonstrated by incorporating and stabilizing adenovirus and MVA vaccines. Clinically-available trivalent inactivated influenza vaccine (TIV) in DMN patches is fully stable for greater than 6months at 40°C. Immunization using low dose TIV-loaded DMN patches induced significantly higher antibody responses compared to intramuscular-based immunization in mice. TIV-loaded patches also induced a broader, heterosubtypic neutralizing antibody response. By addressing issues that will be faced in large-scale fill-finish DMN fabrication processes and demonstrating superior thermostable characteristics and immunogenicity, this study progresses the translation of this microneedle platform to eventual clinical deployment. Copyright © 2016 Elsevier B.V. All rights reserved.

Opportunities and challenges of Web 2.0 for vaccination decisions.

Science.gov (United States)

Betsch, Cornelia; Brewer, Noel T; Brocard, Pauline; Davies, Patrick; Gaissmaier, Wolfgang; Haase, Niels; Leask, Julie; Renkewitz, Frank; Renner, Britta; Reyna, Valerie F; Rossmann, Constanze; Sachse, Katharina; Schachinger, Alexander; Siegrist, Michael; Stryk, Marybelle

2012-05-28

A growing number of people use the Internet to obtain health information, including information about vaccines. Websites that allow and promote interaction among users are an increasingly popular source of health information. Users of such so-called Web 2.0 applications (e.g. social media), while still in the minority, represent a growing proportion of online communicators, including vocal and active anti-vaccination groups as well as public health communicators. In this paper, the authors: define Web 2.0 and examine how it may influence vaccination decisions; discuss how anti-vaccination movements use Web 2.0 as well as the challenges Web 2.0 holds for public health communicators; describe the types of information used in these different settings; introduce the theoretical background that can be used to design effective vaccination communication in a Web 2.0 environment; make recommendations for practice and pose open questions for future research. The authors conclude that, as a result of the Internet and Web 2.0, private and public concerns surrounding vaccinations have the potential to virally spread across the globe in a quick, efficient and vivid manner. Web 2.0 may influence vaccination decisions by delivering information that alters the perceived personal risk of vaccine-preventable diseases or vaccination side-effects. It appears useful for public health officials to put effort into increasing the effectiveness of existing communication by implementing interactive, customized communication. A key step to providing successful public health communication is to identify those who are particularly vulnerable to finding and using unreliable and misleading information. Thus, it appears worthwhile that public health websites strive to be easy to find, easy to use, attractive in its presentation and readily provide the information, support and advice that the searcher is looking for. This holds especially when less knowledgeable individuals are in need of reliable

Peptide vaccination against multiple myeloma using peptides derived from anti-apoptotic protein

DEFF Research Database (Denmark)

Jørgensen, Nicolai Grønne Dahlager; Ahmad, Shamaila Munir; Abildgaard, N.

2016-01-01

The B-cell lymphoma-2 (Bcl-2) family of proteins play a crucial role in multiple myeloma (MM), contributing to lacking apoptosis which is a hallmark of the disease. This makes the Bcl-2 proteins interesting targets for therapeutic peptide vaccination. We report a phase I trial of therapeutic vacc...... vaccination. Vaccination against Bcl-2 was well tolerated and was able to induce immune responses in patients with relapsed MM. © Stem Cell Investigation. All rights reserved.......The B-cell lymphoma-2 (Bcl-2) family of proteins play a crucial role in multiple myeloma (MM), contributing to lacking apoptosis which is a hallmark of the disease. This makes the Bcl-2 proteins interesting targets for therapeutic peptide vaccination. We report a phase I trial of therapeutic...... vaccination with peptides from the proteins Bcl-2, Bcl-XL and Mcl-1 in patients with relapsed MM. Vaccines were given concomitant with bortezomib. Out of 7 enrolled patients, 4 received the full course of 8 vaccinations. The remaining 3 patients received fewer vaccinations due to progression, clinical...

Phase 2 Placebo-Controlled Trial of Two Vaccines to Prevent Ebola in Liberia.

Science.gov (United States)

Kennedy, Stephen B; Bolay, Fatorma; Kieh, Mark; Grandits, Greg; Badio, Moses; Ballou, Ripley; Eckes, Risa; Feinberg, Mark; Follmann, Dean; Grund, Birgit; Gupta, Swati; Hensley, Lisa; Higgs, Elizabeth; Janosko, Krisztina; Johnson, Melvin; Kateh, Francis; Logue, James; Marchand, Jonathan; Monath, Thomas; Nason, Martha; Nyenswah, Tolbert; Roman, François; Stavale, Eric; Wolfson, Julian; Neaton, James D; Lane, H Clifford

2017-10-12

The safety and efficacy of vaccines to prevent Ebola virus disease (EVD) were unknown when the incidence of EVD was peaking in Liberia. We initiated a randomized, placebo-controlled, phase 3 trial of the chimpanzee adenovirus 3 vaccine (ChAd3-EBO-Z) and the recombinant vesicular stomatitis virus vaccine (rVSV∆G-ZEBOV-GP) in Liberia. A phase 2 subtrial was embedded to evaluate safety and immunogenicity. Because the incidence of EVD declined in Liberia, the phase 2 component was expanded and the phase 3 component was eliminated. A total of 1500 adults underwent randomization and were followed for 12 months. The median age of the participants was 30 years; 36.6% of the participants were women. During the week after the administration of vaccine or placebo, adverse events occurred significantly more often with the active vaccines than with placebo; these events included injection-site reactions (in 28.5% of the patients in the ChAd3-EBO-Z group and 30.9% of those in the rVSV∆G-ZEBOV-GP group, as compared with 6.8% of those in the placebo group), headache (in 25.1% and 31.9%, vs. 16.9%), muscle pain (in 22.3% and 26.9%, vs. 13.3%), feverishness (in 23.9% and 30.5%, vs. 9.0%), and fatigue (in 14.0% and 15.4%, vs. 8.8%) (PLiberia showed the capability of conducting rigorous research during an outbreak. By 1 month after vaccination, the vaccines had elicited immune responses that were largely maintained through 12 months. (Funded by the National Institutes of Allergy and Infectious Diseases and the Liberian Ministry of Health; PREVAIL I ClinicalTrials.gov number, NCT02344407 .).

Construction and engineering of a thermostable self-sufficient cytochrome P450

Energy Technology Data Exchange (ETDEWEB)

Mandai, Takao; Fujiwara, Shinsuke [Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337 (Japan); Imaoka, Susumu, E-mail: imaoka@kwansei.ac.jp [Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337 (Japan)

2009-06-19

CYP175A1 is a thermophilic cytochrome P450 and hydroxylates {beta}-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP{sup +} reductase (FNR): H{sub 2}N-CYP175A1-Fdx-FNR-COOH (175FR) and H{sub 2}N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V{sub max} value for {beta}-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k{sub m} values of these enzymes were similar. 175RF retained 50% residual activity even at 80 {sup o}C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.

Construction and engineering of a thermostable self-sufficient cytochrome P450

International Nuclear Information System (INIS)

Mandai, Takao; Fujiwara, Shinsuke; Imaoka, Susumu

2009-01-01

CYP175A1 is a thermophilic cytochrome P450 and hydroxylates β-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP + reductase (FNR): H 2 N-CYP175A1-Fdx-FNR-COOH (175FR) and H 2 N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V max value for β-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k m values of these enzymes were similar. 175RF retained 50% residual activity even at 80 o C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.

Thermostable Alcohol Dehydrogenase from Thermococcus kodakarensis KOD1 for Enantioselective Bioconversion of Aromatic Secondary Alcohols

Science.gov (United States)

Wu, Xi; Zhang, Chong; Orita, Izumi; Imanaka, Tadayuki

2013-01-01

A novel thermostable alcohol dehydrogenase (ADH) showing activity toward aromatic secondary alcohols was identified from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkADH). The gene, tk0845, which encodes an aldo-keto reductase, was heterologously expressed in Escherichia coli. The enzyme was found to be a monomer with a molecular mass of 31 kDa. It was highly thermostable with an optimal temperature of 90°C and a half-life of 4.5 h at 95°C. The apparent Km values for the cofactors NAD(P)+ and NADPH were similar within a range of 66 to 127 μM. TkADH preferred secondary alcohols and accepted various ketones and aldehydes as substrates. Interestingly, the enzyme could oxidize 1-phenylethanol and its derivatives having substituents at the meta and para positions with high enantioselectivity, yielding the corresponding (R)-alcohols with optical purities of greater than 99.8% enantiomeric excess (ee). TkADH could also reduce 2,2,2-trifluoroacetophenone to (R)-2,2,2-trifluoro-1-phenylethanol with high enantioselectivity (>99.6% ee). Furthermore, the enzyme showed high resistance to organic solvents and was particularly highly active in the presence of H2O–20% 2-propanol and H2O–50% n-hexane or n-octane. This ADH is expected to be a useful tool for the production of aromatic chiral alcohols. PMID:23354700

Design and Testing of a Thermostable Platform for Multimerization of Single Domain Antibodies

Science.gov (United States)

2012-08-01

H.J. Properties , production, and applications of camelid single domain antibody fragments. Appl. Microbiol. Biot. 2007, 77, 13‒22. 2. Goldman...Conway, J.; Sherwood, L.J.; Fech, M.; Vo, B.; Liu, J.L.; Hayhurst, A. Thermostable llama single domain antibodies for detection of Botulinum A...antiparallel coiled-coil inserted. J. Mol. Bio. 2001, 306, 25‒35. 9. Liu, J.L.; Anderson, G.P.; Goldman, E.R. Isolation of anti- toxin single domain

Vaccination Coverage Among Children Aged 2 Years - U.S. Affiliated Pacific Islands, April-October, 2016.

Science.gov (United States)

Tippins, Ashley; Murthy, Neil; Meghani, Mehreen; Solsman, Amy; Apaisam, Carter; Basilius, Merlyn; Eckert, Maribeth; Judicpa, Peter; Masunu, Yolanda; Pistotnik, Kelsey; Pedro, Daisy; Sasamoto, Jeremy; Underwood, J Michael

2018-05-25

Vaccine-preventable diseases (VPDs) cause substantial morbidity and mortality in the United States Affiliated Pacific Islands (USAPI).* CDC collaborates with USAPI immunization programs to monitor vaccination coverage. In 2016, † USAPI immunization programs and CDC piloted a method for estimating up-to-date status among children aged 2 years using medical record abstraction to ascertain regional vaccination coverage. This was the first concurrent assessment of childhood vaccination coverage across five USAPI jurisdictions (American Samoa; Chuuk State, Federated States of Micronesia [FSM]; Commonwealth of the Northern Mariana Islands [CNMI]; Republic of the Marshall Islands [RMI]; and Republic of Palau). § Differences in vaccination coverage between main and outer islands ¶ were assessed for two jurisdictions where data were adequate.** Series coverage in this report includes the following doses of vaccines: ≥4 doses of diphtheria and tetanus toxoids and acellular pertussis vaccine (DTaP); ≥3 doses of inactivated poliovirus vaccine (IPV); ≥1 dose of measles, mumps, and rubella vaccine (MMR); ≥3 doses of Haemophilus influenzae type B (Hib) vaccine; ≥3 doses of hepatitis B (HepB) vaccine; and ≥4 doses of pneumococcal conjugate vaccine (PCV); i.e., 4:3:1:3:3:4. Coverage with ≥3 doses of rotavirus vaccine was also assessed. Completion of the recommended series of each of these vaccines †† was <90% in all jurisdictions except Palau. Coverage with the full recommended six-vaccine series (4:3:1:3:3:4) ranged from 19.5% (Chuuk) to 69.1% (Palau). In RMI and Chuuk, coverage was lower in the outer islands than in the main islands for most vaccines, with differences ranging from 0.9 to 66.8 percentage points. Medical record abstraction enabled rapid vaccination coverage assessment and timely dissemination of results to guide programmatic decision-making. Effectively monitoring vaccination coverage, coupled with implementation of data-driven interventions

Rational Design of Disulfide Bonds Increases Thermostability of a Mesophilic 1,3-1,4-β-Glucanase from Bacillus terquilensis.

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Chengtuo Niu

Full Text Available 1,3-1,4-β-glucanase is an important biocatalyst in brewing industry and animal feed industry, while its low thermostability often reduces its application performance. In this study, the thermostability of a mesophilic β-glucanase from Bacillus terquilensis was enhanced by rational design and engineering of disulfide bonds in the protein structure. Protein spatial configuration was analyzed to pre-exclude the residues pairs which negatively conflicted with the protein structure and ensure the contact of catalytic center. The changes in protein overall and local flexibility among the wild-type enzyme and the designated mutants were predicted to select the potential disulfide bonds for enhancement of thermostability. Two residue pairs (N31C-T187C and P102C-N125C were chosen as engineering targets and both of them were proved to significantly enhance the protein thermostability. After combinational mutagenesis, the double mutant N31C-T187C/P102C-N125C showed a 48.3% increase in half-life value at 60°C and a 4.1°C rise in melting temperature (Tm compared to wild-type enzyme. The catalytic property of N31C-T187C/P102C-N125C mutant was similar to that of wild-type enzyme. Interestingly, the optimal pH of double mutant was shifted from pH6.5 to pH6.0, which could also increase its industrial application. By comparison with mutants with single-Cys substitutions, the introduction of disulfide bonds and the induced new hydrogen bonds were proved to result in both local and overall rigidification and should be responsible for the improved thermostability. Therefore, the introduction of disulfide bonds for thermostability improvement could be rationally and highly-effectively designed by combination with spatial configuration analysis and molecular dynamics simulation.

Screening of strains with the high activity and thermostability nattokinase by {sup 60}Co γ-ray irradiation

Energy Technology Data Exchange (ETDEWEB)

Shuying, Li; Ying, Nie; Huan, Du; Xuanming, Tang [Institute of Agro-products Processing Science and Technology, Chinese Academy of Agricultural Sciences, Beijing (China); Zhonglin, Zhao [College of Sciences, Henan Agricultural University, Zhengzhou (China); Xin, Ma [Agricultural Information Institute, Chinese Academic of Agricultural Sciences, Beijing (China); Yan, Li [School of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou (China)

2013-06-15

In this study, Bacillus natto was irradiated by {sup 60}Co γ-ray, and activity was determined by Casein plate method in order to get high activity and thermostability strains. 60 strains with high activity were obtained through irradiation by 800 Gy {sup 60}Co γ-ray. In this dose, the positive mutation rate was 45%. Then 60 strains was treated by different tempreture and 11 strains showed thermostability at 65℃. (authors)

Screening of strains with the high activity and thermostability nattokinase by "6"0Co γ-ray irradiation

International Nuclear Information System (INIS)

Li Shuying; Nie Ying; Du Huan; Tang Xuanming; Zhao Zhonglin; Ma Xin; Li Yan

2013-01-01

In this study, Bacillus natto was irradiated by "6"0Co γ-ray, and activity was determined by Casein plate method in order to get high activity and thermostability strains. 60 strains with high activity were obtained through irradiation by 800 Gy "6"0Co γ-ray. In this dose, the positive mutation rate was 45%. Then 60 strains was treated by different tempreture and 11 strains showed thermostability at 65℃. (authors)

Vaccination of dogs with canine parvovirus type 2b (CPV-2b) induces neutralising antibody responses to CPV-2a and CPV-2c.

Science.gov (United States)

Wilson, Stephen; Illambas, Joanna; Siedek, Elisabeth; Stirling, Catrina; Thomas, Anne; Plevová, Edita; Sture, Gordon; Salt, Jeremy

2014-09-22

Since the identification of canine parvovirus type 2, three variants have subsequently been observed differing from the historical CPV-2 and each other by 1-2 amino acids only. As a result there has been considerable research into differential diagnostics, with some researchers indicating there is a need for new vaccines containing different strains of CPV-2. In this study we investigated whether vaccination with a CPV-2b containing vaccine would induce cross-reactive antibody responses to the other CPV-2 variants. Two studies where dogs were vaccinated with a multivalent vaccine, subsequently challenged with CPV-2b and sera samples analysed are presented. Six week old pups with defined serological status were vaccinated twice, three weeks apart and challenged either 5 weeks (MDA override study) or one year after vaccination (duration of immunity study). Sera samples were collected before each vaccination and at periods throughout each study. In each study the antibody profiles were very similar; serological responses against CPV-2a, CPV-2b and CPV-2c were higher than those for CPV-2. Nevertheless, responses against CPV-2 were well above levels considered clinically protective. In each study dogs also showed a rapid increase in antibody titres following vaccination, reached a plateau following second vaccination with a slight decline to challenge after which rapid anamnestic responses were seen. Evaluation of the serological responses suggests vaccination with CPV-2b would cross-protect against CPV-2a and CPV-2c, as well as against CPV-2 which is now extinct in the field. In conclusion we have demonstrated that vaccination of minimum aged dogs with a multivalent vaccine containing the CPV-2b variant strain will induce serological responses which are cross-reactive against all currently circulating field strains, CPV-2a and CPV-2c, and the now extinct field strain CPV-2. Copyright © 2014 Elsevier Ltd. All rights reserved.

[Immunization with Bifidobacterium bifidum-vectored OprI vaccine of Pseudomonas aeruginosa enhances inhibitory effect on P. aeruginosa in mice].

Science.gov (United States)

Liu, Xiao; Li, Wengui

2017-08-01

Objective To study the pulmonary bacterial loads, splenocyte proliferation, distributions of T cell subsets and cell apoptosis in mice immunized with Bifidobacterium bifidum-vectored OprI (Bb-OprI) vaccine of Pseudomonas aeruginosa and challenged with P. aeruginosa PA01 strain. Methods BALB/c mice were immunized with 5×10 9 CFUs of vaccine by intragastric administration, 3 times a week for 3 weeks, and challenged intranasally with 5×10 6 CFUs of PA01 strain at the fourth week after the first immunization. At the second week after the challenge, all mice were sacrificed to separate their lungs and spleens, and the pulmonary bacterial loads were counted. The proliferation of the splenocytes was determined by MTT assay. The splenic CD4 + , CD8 + T cell subsets and the apoptotic rate of splenocytes were detected by flow cytometry. Results The number of pulmonary bacterial colonies in the mice immunized with the vaccine and challenged with PA01 strain decreased, while the proliferation of splenocytes and the proportion of CD4 + T cells markedly increased, and the apoptosis of splenocytes was notably reduced. Conclusion The intragastric vaccination of recombinant Bb-OprI vaccine can increase the proportion of CD4 + T cells and enhance the inhibitory effect on P. aeruginosa.

Cooperation between catalytic and DNA binding domains enhances thermostability and supports DNA synthesis at higher temperatures by thermostable DNA polymerases.

Science.gov (United States)

Pavlov, Andrey R; Pavlova, Nadejda V; Kozyavkin, Sergei A; Slesarev, Alexei I

2012-03-13

We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases [Pavlov, A. R., et al. (2002) Proc. Natl. Acad. Sci. U.S.A.99, 13510-13515]. The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various sequence-nonspecific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting helix-hairpin-helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of Topo V HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105 °C by maintaining processivity of DNA synthesis at high temperatures. We found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding of templates to DNA polymerases.

Cooperation between Catalytic and DNA-binding Domains Enhances Thermostability and Supports DNA Synthesis at Higher Temperatures by Thermostable DNA Polymerases

Science.gov (United States)

Pavlov, Andrey R.; Pavlova, Nadejda V.; Kozyavkin, Sergei A.; Slesarev, Alexei I.

2012-01-01

We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases (Pavlov et. al., (2002) Proc. Natl. Acad. Sci. USA 99, 13510–13515). The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various non-specific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting Helix-hairpin-Helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species, but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of TopoV HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105°C by maintaining processivity of DNA synthesis at high temperatures. We also found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding templates to DNA polymerases. PMID:22320201

Vaccination with p53-peptide-pulsed dendritic cells, of patients with advanced breast cancer: report from a phase I study

DEFF Research Database (Denmark)

Svane, Inge Marie; Pedersen, Anders E; Johnsen, Hans E

2004-01-01

the treatment. In conclusion, the strategy for p53-DC vaccination seems safe and without toxicity. Furthermore, indications of both immunologic and clinical effect were found in heavily pretreated patients with advanced breast cancer. An independent clinical effect of repeated administration of DCs and IL-2 can......Peptides derived from over-expressed p53 protein are presented by class I MHC molecules and may act as tumour-associated epitopes. Due to the diversity of p53 mutations, immunogenic peptides representing wild-type sequences are preferable as a basis for a broad-spectrum p53-targeting cancer vaccine......) loaded with a cocktail of three wild-type and three modified p53 peptides are being analysed in six HLA-A2+ patients with progressive advanced breast cancer. Vaccinations were well tolerated and no toxicity was observed. Disease stabilisation was seen in two of six patients, one patient had a transient...

Vaccination with human papillomavirus pseudovirus-encapsidated plasmids targeted to skin using microneedles.

Directory of Open Access Journals (Sweden)

Rhonda C Kines

Full Text Available Human papilloma virus-like particles (HPV VLP serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from respiratory syncytial virus (RSV in HPV pseudovirions (PsV is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles. In this study we asked: 1 whether HPV16 VLP administered by microneedles could induce protective immune responses to HPV16 and 2 whether HPV16 PsV-encapsidated plasmids delivered by microneedles could elicit immune responses to both HPV and the antigen delivered by the transgene. Mice immunized with HPV16 VLP coated microneedles generated robust neutralizing antibody responses and were protected from HPV16 challenge. Microneedle arrays coated with HPV16-M/M2 or HPV16-F protein (genes of RSV were then tested and dose-dependent HPV and F-specific antibody responses were detected post-immunization, and M/M2-specific T-cell responses were detected post RSV challenge, respectively. HPV16 PsV-F immunized mice were fully protected from challenge with HPV16 PsV and had reduced RSV viral load in lung and nose upon intranasal RSV challenge. In summary, HPV16 PsV-encapsidated DNA delivered by microneedles induced neutralizing antibody responses against HPV and primed for antibody and T-cell responses to RSV antigens encoded by the encapsidated plasmids. Although the immunogenicity of the DNA component was just above the dose response threshold, the HPV-specific immunity was robust. Taken together, these data suggest microneedle delivery of lyophilized HPV PsV could provide a practical, thermostable combined vaccine approach that could be developed for clinical evaluation.

Clinical trial to evaluate safety and immunogenicity of an oral inactivated enterotoxigenic Escherichia coli prototype vaccine containing CFA/I overexpressing bacteria and recombinantly produced LTB/CTB hybrid protein.

Science.gov (United States)

Lundgren, A; Leach, S; Tobias, J; Carlin, N; Gustafsson, B; Jertborn, M; Bourgeois, L; Walker, R; Holmgren, J; Svennerholm, A-M

2013-02-06

We have developed a new oral vaccine against enterotoxigenic Escherichia coli (ETEC) diarrhea containing killed recombinant E. coli bacteria expressing increased levels of ETEC colonization factors (CFs) and a recombinant protein (LCTBA), i.e. a hybrid between the binding subunits of E. coli heat labile toxin (LTB) and cholera toxin (CTB). We describe a randomized, comparator controlled, double-blind phase I trial in 60 adult Swedish volunteers of a prototype of this vaccine. The safety and immunogenicity of the prototype vaccine, containing LCTBA and an E. coli strain overexpressing the colonization factor CFA/I, was compared to a previously developed oral ETEC vaccine, consisting of CTB and inactivated wild type ETEC bacteria expressing CFA/I (reference vaccine). Groups of volunteers were given two oral doses of either the prototype or the reference vaccine; the prototype vaccine was administered at the same or a fourfold higher dosage than the reference vaccine. The prototype vaccine was found to be safe and equally well-tolerated as the reference vaccine at either dosage tested. The prototype vaccine induced mucosal IgA (fecal secretory IgA and intestine-derived IgA antibody secreting cell) responses to both LTB and CFA/I, as well as serum IgA and IgG antibody responses to LTB. Immunization with LCTBA resulted in about twofold higher mucosal and systemic IgA responses against LTB than a comparable dose of CTB. The higher dose of the prototype vaccine induced significantly higher fecal and systemic IgA responses to LTB and fecal IgA responses to CFA/I than the reference vaccine. These results demonstrate that CF over-expression and inclusion of the LCTBA hybrid protein in an oral inactivated ETEC vaccine does not change the safety profile when compared to a previous generation of such a vaccine and that the prototype vaccine induces significant dose dependent mucosal immune responses against CFA/I and LTB. Copyright © 2013 Elsevier Ltd. All rights reserved.

Epidemiological impact of a genital herpes type 2 vaccine for young females.

Directory of Open Access Journals (Sweden)

Yijun Lou

Full Text Available Genital Herpes, which is caused by Herpes Simplex Virus-1 or -2 (HSV-1, -2, predominantly HSV-2 is a sexually transmitted infection (STI that causes a chronic latent infection with outbreak episodes linked to transmission. Antiviral therapies are effective in reducing viral shedding during these episodes, but are ineffective as a whole since many outbreaks are asymptomatic or have mild symptoms. Thus, the development of a vaccine for genital herpes is needed to control this disease. The question of how to implement such a vaccine program is an important one, and may be similar to the vaccination program for Human Papilloma Virus (HPV for young females. We have developed a mathematical model to describe the epidemiology of vaccination targeting young females against HSV-2. The model population is delineated with respect to age group, sexual activity and infection status including oral infection of HSV-1, which may affect vaccine efficacy. A threshold parameter R(C, which determines the level of vaccine uptake needed to eradicate HSV-2, is found. Computer simulation shows that an adolescent-only vaccination program may be effective in eliminating HSV-2 disease, however, the success of extinction greatly depends on the level of vaccine uptake, the vaccine efficacy, the age of sexual maturity and safe sex practices. However, the time course of eradication would take many years. We also investigate the prevalence of infection in the total population and in women between 16-30 years of age before and after vaccination has been introduced, and show that the adolescent-only vaccination program can be effective in reducing disease prevalence in these populations depending on the level of vaccine uptake and vaccine efficacy. This will also result in a decrease of maternal-fetal transmission of HSV-2 infection. Another important, if commonsense, conclusion is that vaccination of some females reduces infection in men, which then reduces infection in women.

Evaluation of the thermal stability of a novel strain of live-attenuated mumps vaccine (RS-12 strain) lyophilized in different stabilizers.

Science.gov (United States)

Jamil, Razieh Kamali; Taqavian, Mohammad; Sadigh, Zohreh-Azita; Shahkarami, Mohammad-Kazem; Esna-Ashari, Fatemeh; Hamkar, Rasool; Hosseini, Seyedeh-Marzieh; Hatami, Alireza

2014-04-01

The stability of live-attenuated viral vaccines is important for immunization efficacy. Here, the thermostabilities of lyophilized live-attenuated mumps vaccine formulations in two different stabilizers, a trehalose dihydrate-based stabilizer and a stabilizer containing sucrose, human serum albumin and sorbitol were investigated using accelerated stability tests at 4°C, 25°C and 37°C at time points between 4h (every 4h for the first 24h) and 1 week. Even under the harshest storage conditions of 37°C for 1 week, the 50% cell culture infective dose (CCID50) determined from titrations in Vero cells dropped by less than 10-fold using each stabilizer formulation and thus complied with the World Health Organization's requirements for the potency of live-attenuated mumps vaccines. However, as the half-life of the RS-12 strain mumps virus infectivity was lengthened substantially at elevated temperatures using the trehalose dihydrate (TD)-based stabilizer, this stabilizer is recommended for vaccine use. Copyright © 2013 Elsevier B.V. All rights reserved.

The European I-MOVE Multicentre 2013-2014 Case-Control Study. Homogeneous moderate influenza vaccine effectiveness against A(H1N1)pdm09 and heterogenous results by country against A(H3N2).

LENUS (Irish Health Repository)

Valenciano, Marta

2015-06-04

In the first five I-MOVE (Influenza Monitoring Vaccine Effectiveness in Europe) influenza seasons vaccine effectiveness (VE) results were relatively homogenous among participating study sites. In 2013-2014, we undertook a multicentre case-control study based on sentinel practitioner surveillance networks in six European Union (EU) countries to measure 2013-2014 influenza VE against medically-attended influenza-like illness (ILI) laboratory-confirmed as influenza. Influenza A(H3N2) and A(H1N1)pdm09 viruses co-circulated during the season.

Systems of Rb2I2-CdI2-PbI2 and Cs2I2-CdI2-PbI2

International Nuclear Information System (INIS)

Volchanskaya, V.V.; Il'yasov, I.I.

1979-01-01

The Rb 2 I 2 -CdI 2 -PbI 2 and Cs 2 I 2 -CdI 2 -PbI 2 triple systems have been studied, using the visual-polythermal method. The liquidus of the systems researched consists of the components and compounds crystallization fields: 2RbIxCdI 2 , 2RbIxRbI 2 , RbIxPbI 2 and 2CsIxCdI 2 , 4CsIxPbI 2 , CsIxPbI 2 , respectively. The crystallization fields converge in four non-variant points at 360, 280, 205 and 192 deg C in the Rb 2 I 2 -CdI 2 -PbI 2 system and at 375, 368, 208 and 190 deg C in the CsI 2 -CdI 2 -PbI 2 system

Antibody persistence 5 years after vaccination at 2 to 10 years of age with Quadrivalent MenACWY-CRM conjugate vaccine, and responses to a booster vaccination.

Science.gov (United States)

Block, Stan L; Christensen, Shane; Verma, Bikash; Xie, Fang; Keshavan, Pavitra; Dull, Peter M; Smolenov, Igor

2015-04-27

In a multi-center extension study, children 2-10 years of age, initially vaccinated with one or two doses (2-5 year-olds) or one dose (6-10 year-olds) of quadrivalent meningococcal CRM197-conjugate vaccine (MenACWY-CRM), were assessed five years later for antibody persistence and booster response using serum bactericidal assay with human complement (hSBA). Children 7-10 and 11-15 years of age, who received MenACWY-CRM in the original study, and age-matched vaccine-naïve children, were enrolled in this extension study. After an initial blood draw, children received one dose of MenACWY-CRM as booster or primary dose, with a second blood draw 28 days later. hSBA titers decreased five years after primary vaccination, but were higher than in non-vaccinated controls against serogroups C, W and Y, with substantial proportions having titers ≥8: 7-22% for A, 32-57% for C, 74-83% for W, and 48-54% for Y. Previously-vaccinated children demonstrated booster responses to revaccination against all four serogroups. Responses to primary vaccination in vaccine-naïve controls were lower and similar to primary responses observed in the original study. All vaccinations were generally well tolerated, with no safety concern raised. Approximately half the children vaccinated as 2-10 year-olds maintained protective antibodies against serogroups C, W and Y five years later, but fewer did against serogroup A. Declining titers five years after vaccination and robust booster responses suggest that five years may be an appropriate interval to revaccinate children, subject to epidemiology and delivery considerations. Copyright © 2015 Elsevier Ltd. All rights reserved.

Vaccination with canine parvovirus type 2 (CPV-2) protects against challenge with virulent CPV-2b and CPV-2c.

Science.gov (United States)

Siedek, Elisabeth M; Schmidt, Holger; Sture, Gordon H; Raue, Rüdiger

2011-01-01

Mutations in canine parvovirus (CPV) field isolates have created concerns regarding the ability of vaccines containing CPV-2 to protect against infection with the newly identified antigenic types CPV-2b and CPV-2c. To address this concern, the efficacy of CPV-2 strain NL-35-D currently in use as a commercial vaccine was demonstrated against an oral challenge with CPV-2b and CPV-2c, respectively. Clinically healthy specific pathogen free Beagle dogs were either vaccinated or treated with water for injection first at 8-9 weeks of age and again at 11-12 weeks of age. All dogs were challenged either with CPV-2b or CPV-2c three weeks after the second vaccination. During the two week period following challenge, clinical signs, white blood cell counts, serology by haemagglutination inhibition (HI) and serum neutralisation tests, and virus shedding by haemagglutination test were assessed. All control dogs developed clinical signs of parvovirosis (including pyrexia and leucopenia) and shed virus. Vaccinated dogs seroconverted (HI titres > or =80), remained healthy throughout the study and shed more than 100 times less virus than controls. In conclusion, vaccination with the low passage, high titre CPV-2 strain NL-35-D cross-protects dogs against virulent challenges with CPV-2b or CPV-2c by preventing disease and substantially reducing viral shedding.

Thermostability and photophysical properties of mixed-ligand carboxylate/benzimidazole Zn(II)-coordination polymers

International Nuclear Information System (INIS)

Barros, Bráulio Silva; Chojnacki, Jaroslaw; Macêdo Soares, Antonia Alice; Kulesza, Joanna; Lourenço da Luz, Leonis; Júnior, Severino Alves

2015-01-01

The reaction between Zn(NO 3 ) 2 ·6H 2 O or Zn(CH 3 COO) 2 ·2H 2 O and isophthalic acid (1,3-H 2 bdc) in the presence of benzimidazole (Hbzim) in dimethylformamide (DMF)/ethanol (EtOH)/H 2 O solvent mixture at room temperature yielded two structurally different coordination polymers: [Zn 2 (1,3-bdc) 2 (Hbzim) 2 ] (1) and [Zn 2 (1,3-bdc)(bzim) 2 ] (2). (1) is a 2D-layered framework with a molecule of benzimidazole coordinated to the Zn center, whereas (2) is a 3D framework with benzimidazolate species acting as a co-ligand and bridging two Zn(II) ions. Reactions performed at 90 °C led to the formation of coordination polymers structurally similar to (2), independently of the Zn(II) source used. In the absence of benzimidazole, the reaction between ZnAc 2 .2H 2 O and 1,3-H 2 bdc at 90 °C resulted in the formation of (3), a 3D coordination polymer Zn(HCOO) 3 (Me 2 NH 2 + ). It was observed that the thermostability and the photophysical properties of (1) and (2) are strongly dependent on the coordination modes and packing of benzimidazole in the solid state. These materials present photoluminescence in the wide range of the spectrum, from UV to IR. A full understanding of a physical process occurring in these intriguing systems, including complete energy level diagrams with possible transitions were provided. - Graphical abstract: Display Omitted - Highlights: • Structurally different Zn(II)-coordination polymers were prepared. • The formation of frameworks was counter anion and temperature dependent. • Photoluminescence in the wide range of the spectrum, from UV to IR was observed. • Thermostability and luminescence depended on bzim packing in the structure

Type I error probability spending for post-market drug and vaccine safety surveillance with binomial data.

Science.gov (United States)

Silva, Ivair R

2018-01-15

Type I error probability spending functions are commonly used for designing sequential analysis of binomial data in clinical trials, but it is also quickly emerging for near-continuous sequential analysis of post-market drug and vaccine safety surveillance. It is well known that, for clinical trials, when the null hypothesis is not rejected, it is still important to minimize the sample size. Unlike in post-market drug and vaccine safety surveillance, that is not important. In post-market safety surveillance, specially when the surveillance involves identification of potential signals, the meaningful statistical performance measure to be minimized is the expected sample size when the null hypothesis is rejected. The present paper shows that, instead of the convex Type I error spending shape conventionally used in clinical trials, a concave shape is more indicated for post-market drug and vaccine safety surveillance. This is shown for both, continuous and group sequential analysis. Copyright © 2017 John Wiley & Sons, Ltd.

Immunisation against PCV2 structural protein by DNA vaccination of mice

DEFF Research Database (Denmark)

Kamstrup, Søren; Barfoed, Annette Malene; Frimann, Tine

2004-01-01

the capsid protein of PCV2 was cloned in a DNA vaccination plasmid and expression of capsid protein was demonstrated in vitro. Mice were gene gun vaccinated three timesand all mice responded serologically by raising antibodies against PCV2. The results suggest, that DNA based vaccination might offer...

Safety and preliminary immunogenicity of Cuban pneumococcal conjugate vaccine candidate in healthy children: a randomized phase I clinical trial.

Science.gov (United States)

Dotres, Carlos P; Puga, Rinaldo; Ricardo, Yariset; Broño, Carmen R; Paredes, Beatriz; Echemendía, Vladimir; Rosell, Sandra; González, Nadezhda; García-Rivera, Dagmar; Valdés, Yury; Goldblatt, David; Vérez-Bencomo, Vicente

2014-09-15

A new heptavalent conjugate vaccine (PCV7-TT) is under development in Cuba. PCV7-TT contains 2 μg of serotypes 1, 5, 14, 18C, 19F, 23F and 4 μg of 6B, each one conjugated to tetanus toxoid (TT). This vaccine was designed with the serotypes that cause most invasive pneumococcal diseases (IPD) worldwide. In the present study, we investigated the safety and explored the immunogenicity of PCV7-TT during a controlled, randomized and double blind clinical trial phase I in 4-5-year-old children. PCV7-TT was well tolerated and as safe as Synflorix used as control vaccine. Following a single-dose vaccination, all individual serotypes included in PCV7-TT induced statistically significant increase of IgG GMC and OPA GMT. These are the first clinical results of PCV7-TT in children and they pave the way toward next clinical trials in children and infants. This clinical trial was published in the Cuban Public Register of Clinical Trials with code RPCEC00000173. Copyright © 2014 Elsevier Ltd. All rights reserved.

Safety, immune and clinical responses in metastatic melanoma patients vaccinated with a long peptide derived from indoleamine 2,3-dioxygenase in combination with ipilimumab

DEFF Research Database (Denmark)

Bjørn, Jon; Iversen, Trine Zeeberg; Nitschke, Nikolaj Juul

2016-01-01

antibody ipilimumab (ipi). METHODS: Ten patients with metastatic melanoma participated in a phase I first-in-human clinical study assessing safety of combining ipi with a 21-mer synthetic peptide vaccine from IDO denoted IDOlong. Secondary and tertiary end points included vaccine and clinical response......BACKGROUND AIM: Indoleamine 2,3-dioxygenase (IDO) is an emerging new target in cancer therapy that can be targeted with active immunotherapy (e.g. through peptide vaccination). Furthermore, IDO has been identified as a key mechanism underlying resistance to treatment with the checkpoint blocking....... RESULTS: Treatment was generally safe and well tolerated. Vaccine related adverse reactions included grade I and II erythema, oedema and pruritus at the vaccination site, which were manageable with mild topical corticosteroids. One patient developed presumed ipi-induced colitis. It initially responded...

Crystallization and X-ray structure analysis of a thermostable penicillin G acylase from Alcaligenes faecalis

International Nuclear Information System (INIS)

Varshney, Nishant Kumar; Suresh Kumar, R.; Ignatova, Zoya; Prabhune, Asmita; Pundle, Archana; Dodson, Eleanor; Suresh, C. G.

2012-01-01

A thermostable penicillin G acylase from A. faecalis has been crystallized in two space groups: C222 1 and P4 1 2 1 2. X-ray diffraction data were collected to 3.3 and 3.5 Å resolution, respectively. The enzyme penicillin G acylase (EC 3.5.1.11) catalyzes amide-bond cleavage in benzylpenicillin (penicillin G) to yield 6-aminopenicillanic acid, an intermediate chemical used in the production of semisynthetic penicillins. A thermostable penicillin G acylase from Alcaligenes faecalis (AfPGA) has been crystallized using the hanging-drop vapour-diffusion method in two different space groups: C222 1 , with unit-cell parameters a = 72.9, b = 86.0, c = 260.2 Å, and P4 1 2 1 2, with unit-cell parameters a = b = 85.6, c = 298.8 Å. Data were collected at 293 K and the structure was determined using the molecular-replacement method. Like other penicillin acylases, AfPGA belongs to the N-terminal nucleophilic hydrolase superfamily, has undergone post-translational processing and has a serine as the N-terminal residue of the β-chain. A disulfide bridge has been identified in the structure that was not found in the other two known penicillin G acylase structures. The presence of the disulfide bridge is perceived to be one factor that confers higher stability to this enzyme

Safety and immunogenicity of rVSVΔG-ZEBOV-GP Ebola vaccine in adults and children in Lambaréné, Gabon: A phase I randomised trial

NARCIS (Netherlands)

Agnandji, Selidji T.; Fernandes, José F.; Bache, Emmanuel B.; Obiang Mba, Régis M.; Brosnahan, Jessica S.; Kabwende, Lumeka; Pitzinger, Paul; Staarink, Pieter; Massinga-Loembe, Marguerite; Krähling, Verena; Biedenkopf, Nadine; Fehling, Sarah Katharina; Strecker, Thomas; Clark, David J.; Staines, Henry M.; Hooper, Jay W.; Silvera, Peter; Moorthy, Vasee; Kieny, Marie-Paule; Adegnika, Akim A.; Grobusch, Martin P.; Becker, Stephan; Ramharter, Michael; Mordmüller, Benjamin; Lell, Bertrand; Krishna, Sanjeev; Kremsner, Peter G.

2017-01-01

The rVSVΔG-ZEBOV-GP vaccine prevented Ebola virus disease when used at 2 × 107 plaque-forming units (PFU) in a trial in Guinea. This study provides further safety and immunogenicity data. A randomised, open-label phase I trial in Lambaréné, Gabon, studied 5 single intramuscular vaccine doses of 3 ×

A defense of compulsory vaccination.

Science.gov (United States)

Flanigan, Jessica

2014-03-01

Vaccine refusal harms and risks harming innocent bystanders. People are not entitled to harm innocents or to impose deadly risks on others, so in these cases there is nothing to be said for the right to refuse vaccination. Compulsory vaccination is therefore justified because non-vaccination can rightly be prohibited, just as other kinds of harmful and risky conduct are rightly prohibited. I develop an analogy to random gunfire to illustrate this point. Vaccine refusal, I argue, is morally similar to firing a weapon into the air and endangering innocent bystanders. By re-framing vaccine refusal as harmful and reckless conduct my aim is to shift the focus of the vaccine debate from non-vaccinators' religious and refusal rights to everyone else's rights against being infected with contagious illnesses. Religious freedom and rights of informed consent do not entitle non-vaccinators to harm innocent bystanders, and so coercive vaccination requirements are permissible for the sake of the potential victims of the anti-vaccine movement.

DIVA vaccine properties of the live chimeric pestivirus strain CP7_E2gif.

Science.gov (United States)

von Rosen, Tanya; Rangelova, Desislava; Nielsen, Jens; Rasmussen, Thomas Bruun; Uttenthal, Åse

2014-06-04

Live modified vaccines to protect against classical swine fever virus (CSFV), based on chimeric pestiviruses, have been developed to enable serological Differentiation of Infected from Vaccinated Animals (DIVA). In this context, the chimeric virus CP7_E2gif vaccine candidate is unique as it does not include any CSFV components. In the present study, the DIVA vaccine properties of CP7_E2gif were evaluated in comparison to the conventional live attenuated Riemser C-strain vaccine. Sera and tonsil samples obtained from pigs immunised with these two vaccines were analysed. No viral RNA was found in serum after vaccination with CP7_E2gif, whereas some serum samples from C-strain vaccinated animals were positive. In both vaccinated groups, individual viral RNA-positive tonsil samples were detected in animals euthanised between 7 and 21 days post vaccination. Furthermore, serum samples from these animals, together with archival samples from pigs vaccinated with CP7_E2gif and subsequently CSFV challenged, were analysed for specific antibodies using ELISAs and for homologous neutralising antibodies. In animals vaccinated with CP7_E2gif, neutralising antibodies were detected from day 10. However, the sera remained negative for anti-CSFV E2-specific antibodies whereas pigs vaccinated with C-strain seroconverted against CSFV by 14 days after vaccination, as determined by a CSFV-E2 specific blocking ELISA. One week after subsequent CSFV challenge, a strong anti-CSFV E2 reaction was detected in CP7_E2gif vaccinated pigs and anti-E(rns) antibodies were detected from 10 days after infection. In conclusion, CP7_E2gif has the potential to be used as a DIVA vaccine in combination with detection of anti-CSFV E2-specific antibodies. Copyright © 2014 Elsevier B.V. All rights reserved.

Crystallization and preliminary X-ray study of a family 10 alkali-thermostable xylanase from alkalophilic Bacillus sp. strain NG-27

Energy Technology Data Exchange (ETDEWEB)

Manikandan, K. [Department of Physics, Indian Institute of Science, Bangalore 560 012 (India); Bhardwaj, Amit [International Centre for Genetic Engineering and Biotechnology, New Delhi 110 067 (India); Ghosh, Amit [Institute of Microbial Technology, Sector 39-A, Chandigarh 160 036 (India); Reddy, V. S. [International Centre for Genetic Engineering and Biotechnology, New Delhi 110 067 (India); Ramakumar, S., E-mail: ramak@physics.iisc.ernet.in [Department of Physics, Indian Institute of Science, Bangalore 560 012 (India); Bioinformatics Centre, Indian Institute of Science, Bangalore 560 012 (India)

2005-08-01

A family 10 alkali-thermostable xylanase from Bacillus sp. NG-27 has been crystallized. A diffraction data set has been collected to 2.2 Å resolution. Xylanases (EC 3.2.1.8) catalyze the hydrolysis of β-1,4-glycosidic linkages within xylan, a major hemicellulose component in the biosphere. The extracellular endoxylanase (XylnA) from the alkalophilic Bacillus sp. strain NG-27 belongs to family 10 of the glycoside hydrolases. It is active at 343 K and pH 8.4. Moreover, it has attractive features from the point of view of utilization in the paper pulp, animal feed and baking industries since it is an alkali-thermostable protein. In this study, XylnA was purified from the native host source and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 174.5, b = 54.7, c = 131.5 Å, β = 131.2°, and diffract to better than 2.2 Å resolution.

Crystallization and preliminary X-ray study of a family 10 alkali-thermostable xylanase from alkalophilic Bacillus sp. strain NG-27

International Nuclear Information System (INIS)

Manikandan, K.; Bhardwaj, Amit; Ghosh, Amit; Reddy, V. S.; Ramakumar, S.

2005-01-01

A family 10 alkali-thermostable xylanase from Bacillus sp. NG-27 has been crystallized. A diffraction data set has been collected to 2.2 Å resolution. Xylanases (EC 3.2.1.8) catalyze the hydrolysis of β-1,4-glycosidic linkages within xylan, a major hemicellulose component in the biosphere. The extracellular endoxylanase (XylnA) from the alkalophilic Bacillus sp. strain NG-27 belongs to family 10 of the glycoside hydrolases. It is active at 343 K and pH 8.4. Moreover, it has attractive features from the point of view of utilization in the paper pulp, animal feed and baking industries since it is an alkali-thermostable protein. In this study, XylnA was purified from the native host source and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 174.5, b = 54.7, c = 131.5 Å, β = 131.2°, and diffract to better than 2.2 Å resolution

Immune Responses and Protection against Experimental Brucella suis biovar 1 Challenge in Non-vaccinated or RB51-Vaccinated Cattle

Science.gov (United States)

Twenty Hereford heifers, approximately 9 months of age, were vaccinated with saline (control) or 2 x 10**10 CFU of Brucella abortus strain RB51 (RB51) vaccine. Immunologic responses after inoculation demonstrated significantly greater (P<0.05) antibody and proliferative responses to RB51 antigens i...

Glycine in the conserved motif III modulates the thermostability and oxidative stress resistance of peptide deformylase in Mycobacterium tuberculosis.

Science.gov (United States)

Narayanan, Sai Shyam; Sokkar, Pandian; Ramachandran, Murugesan; Nampoothiri, Kesavan Madhavan

2011-07-01

Peptide deformylase (PDF) catalyses the removal of the N-formyl group from the nascent polypeptide during protein maturation. The PDF of Mycobacterium tuberculosis H37Rv (MtbPDF), overexpressed and purified from Escherichia coli, was characterized as an iron-containing enzyme with stability towards H(2) O(2) and moderate thermostability. Substitution of two conserved residues (G49 and L107) from MtbPDF with the corresponding residues found in human PDF affected its deformylase activity. Among characterized PDFs, glycine (G151) in motif III instead of conserved aspartate is characteristic of M. tuberculosis. Although the G151D mutation in MtbPDF increased its deformylase activity and thermostability, it also affected enzyme stability towards H(2) O(2) . Molecular dynamics and docking results confirmed improved substrate binding and catalysis for the G151D mutant and the study provides another possible molecular basis for the stability of MtbPDF against oxidizing agents. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Vaccine decision-making begins in pregnancy: Correlation between vaccine concerns, intentions and maternal vaccination with subsequent childhood vaccine uptake.

Science.gov (United States)

Danchin, M H; Costa-Pinto, J; Attwell, K; Willaby, H; Wiley, K; Hoq, M; Leask, J; Perrett, K P; O'Keefe, Jacinta; Giles, M L; Marshall, H

2017-08-12

Maternal and childhood vaccine decision-making begins prenatally. Amongst pregnant Australian women we aimed to ascertain vaccine information received, maternal immunisation uptake and attitudes and concerns regarding childhood vaccination. We also aimed to determine any correlation between a) intentions and concerns regarding childhood vaccination, (b) concerns about pregnancy vaccination, (c) socioeconomic status (SES) and (d) uptake of influenza and pertussis vaccines during pregnancy and routine vaccines during childhood. Women attending public antenatal clinics were recruited in three Australian states. Surveys were completed on iPads. Follow-up phone surveys were done three to six months post delivery, and infant vaccination status obtained via the Australian Childhood Immunisation Register (ACIR). Between October 2015 and March 2016, 975 (82%) of 1184 mothers consented and 406 (42%) agreed to a follow up survey, post delivery. First-time mothers (445; 49%) had significantly more vaccine concerns in pregnancy and only 73% had made a decision about childhood vaccination compared to 89% of mothers with existing children (p-valuepost delivery survey, 46% and 82% of mothers reported receiving pregnancy influenza and pertussis vaccines respectively. The mother's degree of vaccine hesitancy and two attitudinal factors were correlated with vaccine uptake post delivery. There was no association between reported maternal vaccine uptake or SES and childhood vaccine uptake. First time mothers are more vaccine hesitant and undecided about childhood vaccination, and only two thirds of all mothers believed they received enough information during pregnancy. New interventions to improve both education and communication on childhood and maternal vaccines, delivered by midwives and obstetricians in the Australian public hospital system, may reduce vaccine hesitancy for all mothers in pregnancy and post delivery, particularly first-time mothers. Copyright © 2017 Elsevier Ltd

Thermostability promotes the cooperative function of split adenylate kinases.

Science.gov (United States)

Nguyen, Peter Q; Liu, Shirley; Thompson, Jeremy C; Silberg, Jonathan J

2008-05-01

Proteins can often be cleaved to create inactive polypeptides that associate into functional complexes through non-covalent interactions, but little is known about what influences the cooperative function of the ensuing protein fragments. Here, we examine whether protein thermostability affects protein fragment complementation by characterizing the function of split adenylate kinases from the mesophile Bacillus subtilis (AKBs) and the hyperthermophile Thermotoga neapolitana (AKTn). Complementation studies revealed that the split AKTn supported the growth of Escherichia coli with a temperature-sensitive AK, but not the fragmented AKBs. However, weak complementation occurred when the AKBs fragments were fused to polypeptides that strongly associate, and this was enhanced by a Q16L mutation that thermostabilizes the full-length protein. To examine how the split AK homologs differ in structure and function, their catalytic activity, zinc content, and circular dichroism spectra were characterized. The reconstituted AKTn had higher levels of zinc, greater secondary structure, and >10(3)-fold more activity than the AKBs pair, albeit 17-fold less active than full-length AKTn. These findings provide evidence that the design of protein fragments that cooperatively function can be improved by choosing proteins with the greatest thermostability for bisection, and they suggest that this arises because hyperthermophilic protein fragments exhibit greater residual structure compared to their mesophilic counterparts.

Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-beta-mannosidase from Aspergillus niger BK01.

Science.gov (United States)

Do, Bien-Cuong; Dang, Thi-Thu; Berrin, Jean-Guy; Haltrich, Dietmar; To, Kim-Anh; Sigoillot, Jean-Claude; Yamabhai, Montarop

2009-11-13

Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-beta-mannosidases (1,4-beta-D-mannanases) catalyze the random hydrolysis of beta-1,4-mannosidic linkages in the main chain of beta-mannans. Biodegradation of beta-mannans by the action of thermostable mannan endo-1,4-beta-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS). A gene encoding mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannan mannanohydrolase (E.C. 3.2.1.78), commonly termed beta-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5), was cloned and successfully expressed heterologously (up to 243 microg of active recombinant protein per mL) in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant beta-mannanase is highly thermostable with a half-life time of approximately 56 h at 70 degrees C and pH 4.0. The optimal temperature (10-min assay) and pH value for activity are 80 degrees C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Km values of the enzyme for konjac glucomannan (low viscosity), locust bean gum galactomannan, carob galactomannan (low viscosity), and 1,4-beta-D-mannan (from carob) are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1 and 1.5 mg mL-1, respectively, while the kcat values for these

In Vitro Characterization of Thermostable CAM Rubisco Activase Reveals a Rubisco Interacting Surface Loop1[OPEN

Science.gov (United States)

Shivhare, Devendra

2017-01-01

To maintain metabolic flux through the Calvin-Benson-Bassham cycle in higher plants, dead-end inhibited complexes of Rubisco must constantly be engaged and remodeled by the molecular chaperone Rubisco activase (Rca). In C3 plants, the thermolability of Rca is responsible for the deactivation of Rubisco and reduction of photosynthesis at moderately elevated temperatures. We reasoned that crassulacean acid metabolism (CAM) plants must possess thermostable Rca to support Calvin-Benson-Bassham cycle flux during the day when stomata are closed. A comparative biochemical characterization of rice (Oryza sativa) and Agave tequilana Rca isoforms demonstrated that the CAM Rca isoforms are approximately10°C more thermostable than the C3 isoforms. Agave Rca also possessed a much higher in vitro biochemical activity, even at low assay temperatures. Mixtures of rice and agave Rca form functional hetero-oligomers in vitro, but only the rice isoforms denature at nonpermissive temperatures. The high thermostability and activity of agave Rca mapped to the N-terminal 244 residues. A Glu-217-Gln amino acid substitution was found to confer high Rca activity to rice Rca. Further mutational analysis suggested that Glu-217 restricts the flexibility of the α4-β4 surface loop that interacts with Rubisco via Lys-216. CAM plants thus promise to be a source of highly functional, thermostable Rca candidates for thermal fortification of crop photosynthesis. Careful characterization of their properties will likely reveal further protein-protein interaction motifs to enrich our mechanistic model of Rca function. PMID:28546437

Estimates of pandemic influenza vaccine effectiveness in Europe, 2009-2010: results of Influenza Monitoring Vaccine Effectiveness in Europe (I-MOVE multicentre case-control study.

Directory of Open Access Journals (Sweden)

Marta Valenciano

2011-01-01

Full Text Available BACKGROUND: A multicentre case-control study based on sentinel practitioner surveillance networks from seven European countries was undertaken to estimate the effectiveness of 2009-2010 pandemic and seasonal influenza vaccines against medically attended influenza-like illness (ILI laboratory-confirmed as pandemic influenza A (H1N1 (pH1N1. METHODS AND FINDINGS: Sentinel practitioners swabbed ILI patients using systematic sampling. We included in the study patients meeting the European ILI case definition with onset of symptoms >14 days after the start of national pandemic vaccination campaigns. We compared pH1N1 cases to influenza laboratory-negative controls. A valid vaccination corresponded to >14 days between receiving a dose of vaccine and symptom onset. We estimated pooled vaccine effectiveness (VE as 1 minus the odds ratio with the study site as a fixed effect. Using logistic regression, we adjusted VE for potential confounding factors (age group, sex, month of onset, chronic diseases and related hospitalizations, smoking history, seasonal influenza vaccinations, practitioner visits in previous year. We conducted a complete case analysis excluding individuals with missing values and a multiple multivariate imputation to estimate missing values. The multivariate imputation (n = 2902 adjusted pandemic VE (PIVE estimates were 71.9% (95% confidence interval [CI] 45.6-85.5 overall; 78.4% (95% CI 54.4-89.8 in patients <65 years; and 72.9% (95% CI 39.8-87.8 in individuals without chronic disease. The complete case (n = 1,502 adjusted PIVE were 66.0% (95% CI 23.9-84.8, 71.3% (95% CI 29.1-88.4, and 70.2% (95% CI 19.4-89.0, respectively. The adjusted PIVE was 66.0% (95% CI -69.9 to 93.2 if vaccinated 8-14 days before ILI onset. The adjusted 2009-2010 seasonal influenza VE was 9.9% (95% CI -65.2 to 50.9. CONCLUSIONS: Our results suggest good protection of the pandemic monovalent vaccine against medically attended pH1N1 and no effect of the

Enzimas termoestáveis: fontes, produção e aplicação industrial Thermostable enzymes: sources, production and industrial applications

Directory of Open Access Journals (Sweden)

Eleni Gomes

2007-02-01

Full Text Available REVIEW: Living organisms encountered in hostile environments that are characterized by extreme temperatures rely on novel molecular mechanisms to enhance the thermal stability of their proteins, nucleic acids, lipids and cell membranes. Proteins isolated from thermophilic organisms usually exhibit higher intrinsic thermal stabilities than their counterparts isolated from mesophilic organisms. Although the molecular basis of protein thermostability is only partially understood, structural studies have suggested that the factors that may contribute to enhance protein thermostability mainly include hydrophobic packing, enhanced secondary structure propensity, helix dipole stabilization, absence of residues sensitive to oxidation or deamination, and increased electrostatic interactions. Thermostable enzymes such as amylases, xylanases and pectinases isolated from thermophilic organisms are potentially of interest in the optimization of industrial processes due to their enhanced stability. In the present review, an attempt is made to delineate the structural factors that increase enzyme thermostability and to document the research results in the production of these enzymes.

Evaluation of oral vaccination of village chickens against newcastle ...

African Journals Online (AJOL)

The study was conducted to assess the suitability of soaked parboiled cracked maize as a carrier of I-2 vaccine for oral immunization of village chickens. Chickens were vaccinated once via ocular route and orally with cracked maize at the second and fifth weeks of the experiment. Post vaccination serum was collected 4, 7, ...

Costs and cost-effectiveness of 9-valent human papillomavirus (HPV) vaccination in two East African countries.

Science.gov (United States)

Kiatpongsan, Sorapop; Kim, Jane J

2014-01-01

-effective, the added cost per vaccinated girl could go up to I$5.2 and I$16.2 in the worst-case and best-case scenarios, respectively. At a willingness-to-pay threshold of three times per-capita GDP where the 9-valent vaccine would be considered cost-effective, the thresholds of added costs associated with the 9-valent vaccine were I$27.3, I$14.5 and I$45.3 per vaccinated girl for the base case, worst-case and best-case scenarios, respectively. In Uganda, vaccination with the 9-valent vaccine was very cost-effective when the added cost of the 9-valent vaccine did not exceed I$8.3 per vaccinated girl. To be considered very cost-effective, the added cost per vaccinated girl could go up to I$4.5 and I$13.7 in the worst-case and best-case scenarios, respectively. At a willingness-to-pay threshold of three times per-capita GDP, the thresholds of added costs associated with the 9-valent vaccine were I$23.4, I$12.6 and I$38.4 per vaccinated girl for the base case, worst-case and best-case scenarios, respectively. This study provides a threshold range of incremental costs associated with the 9-valent HPV vaccine that would make it a cost-effective intervention in comparison to currently available HPV vaccines in Kenya and Uganda. These prices represent a 71% and 61% increase over the price offered to the GAVI Alliance ($5 per dose) for the currently available 2- and 4-valent vaccines in Kenya and Uganda, respectively. Despite evidence of cost-effectiveness, critical challenges around affordability and feasibility of HPV vaccination and other competing needs in low-resource settings such as Kenya and Uganda remain.

Useful halophilic, thermostable and ionic liquids tolerant cellulases

Science.gov (United States)

Zhang, Tao; Datta, Supratim; Simmons, Blake A.; Rubin, Edward M.

2016-06-28

The present invention provides for an isolated or recombinant polypeptide comprising an amino acid sequence having at least 70% identity with the amino acid sequence of a Halorhabdus utahensis cellulase, such as Hu-CBH1, wherein said amino acid sequence has a halophilic thermostable and/or thermophilic cellobiohydrolase (CBH) activity. In some embodiments, the polypeptide has a CBH activity that is resistant to up to about 20% of ionic liquids. The present invention also provides for compositions comprising and methods using the isolated or recombinant polypeptide.

Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system.

Science.gov (United States)

Katano, Yuta; Li, Tongyang; Baba, Misato; Nakamura, Miyo; Ito, Masaaki; Kojima, Kenji; Takita, Teisuke; Yasukawa, Kiyoshi

2017-12-01

We attempted to increase the thermostability of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). The eight-site saturation mutagenesis libraries corresponding to Ala70-Arg469 in the whole MMLV RT (Thr24-Leu671), in each of which 1 out of 50 amino acid residues was replaced with other amino acid residue, were constructed. Seven-hundred and sixty eight MMLV RT clones were expressed using a cell-free protein expression system, and their thermostabilities were assessed by the temperature of thermal treatment at which they retained cDNA synthesis activity. One clone D200C was selected as the most thermostable variant. The highest temperature of thermal treatment at which D200C exhibited cDNA synthesis activity was 57ºC, which was higher than for WT (53ºC). Our results suggest that a combination of site saturation mutagenesis library and cell-free protein expression system might be useful for generation of thermostable MMLV RT in a short period of time for expression and selection.

Induction of Type I Interferons by Therapeutic Nanoparticle-Based Vaccination Is Indispensable to Reinforce Cytotoxic CD8+ T Cell Responses During Chronic Retroviral Infection

Science.gov (United States)

Knuschke, Torben; Rotan, Olga; Bayer, Wibke; Kollenda, Sebastian; Dickow, Julia; Sutter, Kathrin; Hansen, Wiebke; Dittmer, Ulf; Lang, Karl S.; Epple, Matthias; Buer, Jan; Westendorf, Astrid M.

2018-01-01

T cell dysfunction and immunosuppression are characteristic for chronic viral infections and contribute to viral persistence. Overcoming these burdens is the goal of new therapeutic strategies to cure chronic infectious diseases. We recently described that therapeutic vaccination of chronic retrovirus infected mice with a calcium phosphate (CaP) nanoparticle (NP)-based vaccine carrier, functionalized with CpG and viral peptides is able to efficiently reactivate the CD8+ T cell response and improve the eradication of virus infected cells. However, the mechanisms underlying this effect were largely unclear. While type I interferons (IFNs I) are considered to drive T cell exhaustion by persistent immune activation during chronic viral infection, we here describe an indispensable role of IFN I induced by therapeutic vaccination to efficiently reinforce cytotoxic CD8+ T cells (CTL) and improve control of chronic retroviral infection. The induction of IFN I is CpG dependent and leads to significant IFN signaling indicated by upregulation of IFN stimulated genes. By vaccinating chronically retrovirus-infected mice lacking the IFN I receptor (IFNAR−/−) or by blocking IFN I signaling in vivo during therapeutic vaccination, we demonstrate that IFN I signaling is necessary to drive full reactivation of CTLs. Surprisingly, we also identified an impaired suppressive capability of regulatory T cells in the presence of IFNα, which implicates an important role for vaccine-induced IFNα in the regulation of the T cell response during chronic retroviral infection. Our data suggest that inducing IFN I signaling in conjunction with the presentation of viral antigens can reactivate immune functions and reduce viral loads in chronic infections. Therefore, we propose CaP NPs as potential therapeutic tool to treat chronic infections. PMID:29740425

Evaluation of protective efficacy of three novel H3N2 canine influenza vaccines.

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Tu, Liqing; Zhou, Pei; Li, Lutao; Li, Xiuzhen; Hu, Renjun; Jia, Kun; Sun, Lingshuang; Yuan, Ziguo; Li, Shoujun

2017-11-17

Canine influenza virus (CIV) has the potential risk to spread in different areas and dog types. Thus, there is a growing need to develop an effective vaccine to control CIV disease. Here, we developed three vaccine candidates: 1) a recombinant pVAX1 vector expressing H3N2 CIV hemagglutinin (pVAX1-HA); 2) a live attenuated canine adenovirus type 2 expressing H3N2 CIV hemagglutinin (rCAV2-HA); and 3) an inactivated H3N2 CIV (A/canine/Guangdong/01/2006 (H3N2)). Mice received an initial intramuscular immunization that followed two booster injections at 2 and 4 weeks post-vaccination (wpv). The splenic lymphocytes were collected to assess the immune responses at 6 wpv. The protective efficacy was evaluated by challenging H3N2 CIV after vaccination (at 6 wpv). Our results demonstrated that all three vaccine candidates elicited cytokine and antibody responses in mice. The rCAV2-HA vaccine and the inactivated vaccine generated efficient protective efficacy in mice, whereas limited protection was provided by the pVAX1-HA DNA vaccine. Therefore, both the rCAV2-HA live recombinant virus and the inactivated CIV could be used as potential novel vaccines against H3N2CIV. This study provides guidance for choosing the most appropriate vaccine for the prevention and control of CIV disease.

Vaccine Adjuvant Incorporation Strategy Dictates Peptide Amphiphile Micelle Immunostimulatory Capacity.

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Zhang, Rui; Kramer, Jake S; Smith, Josiah D; Allen, Brittany N; Leeper, Caitlin N; Li, Xiaolei; Morton, Logan D; Gallazzi, Fabio; Ulery, Bret D

2018-06-01

Current vaccine research has shifted from traditional vaccines (i.e., whole-killed or live-attenuated) to subunit vaccines (i.e., protein, peptide, or DNA) as the latter is much safer due to delivering only the bioactive components necessary to produce a desirable immune response. Unfortunately, subunit vaccines are very weak immunogens requiring delivery vehicles and the addition of immunostimulatory molecules termed adjuvants to convey protective immunity. An interesting type of delivery vehicle is peptide amphiphile micelles (PAMs), unique biomaterials where the vaccine is part of the nanomaterial itself. Due to the modularity of PAMs, they can be readily modified to deliver both vaccine antigens and adjuvants within a singular construct. Through the co-delivery of a model antigenic epitope (Ovalbumin 319-340 -OVA BT ) and a known molecular adjuvant (e.g., 2,3-dipalmitoyl-S-glyceryl cysteine-Pam 2 C), greater insight into the mechanisms by which PAMs can exert immunostimulatory effects was gained. It was found that specific combinations of antigen and adjuvant can significantly alter vaccine immunogenicity both in vitro and in vivo. These results inform fundamental design rules that can be leveraged to fabricate optimal PAM-based vaccine formulations for future disease-specific applications. Graphical Abstract.

Protein thermostability prediction within homologous families using temperature-dependent statistical potentials.

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Fabrizio Pucci

Full Text Available The ability to rationally modify targeted physical and biological features of a protein of interest holds promise in numerous academic and industrial applications and paves the way towards de novo protein design. In particular, bioprocesses that utilize the remarkable properties of enzymes would often benefit from mutants that remain active at temperatures that are either higher or lower than the physiological temperature, while maintaining the biological activity. Many in silico methods have been developed in recent years for predicting the thermodynamic stability of mutant proteins, but very few have focused on thermostability. To bridge this gap, we developed an algorithm for predicting the best descriptor of thermostability, namely the melting temperature Tm, from the protein's sequence and structure. Our method is applicable when the Tm of proteins homologous to the target protein are known. It is based on the design of several temperature-dependent statistical potentials, derived from datasets consisting of either mesostable or thermostable proteins. Linear combinations of these potentials have been shown to yield an estimation of the protein folding free energies at low and high temperatures, and the difference of these energies, a prediction of the melting temperature. This particular construction, that distinguishes between the interactions that contribute more than others to the stability at high temperatures and those that are more stabilizing at low T, gives better performances compared to the standard approach based on T-independent potentials which predict the thermal resistance from the thermodynamic stability. Our method has been tested on 45 proteins of known Tm that belong to 11 homologous families. The standard deviation between experimental and predicted Tm's is equal to 13.6°C in cross validation, and decreases to 8.3°C if the 6 worst predicted proteins are excluded. Possible extensions of our approach are discussed.

Do two current canine parvovirus type 2 and 2b vaccines provide protection against the new type 2c variant?

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Larson, Laurie J; Schultz, R D

2008-01-01

Three groups (n=9 or 10) of 12-week-old canine parvovirus type 2 (CPV-2) antibody-negative puppies were vaccinated: one group with a product containing modified-live CPV-2b (Galaxy DA2PPv; Schering-Plough Animal Health), one group with a product containing modified-live CPV-2 (Continuum DAP, Intervet), and one group (controls) with sterile saline. All puppies receiving CPV-2 and CPV-2b vaccines developed antibody as determined by the hemagglutination inhibition assay. All groups of puppies were challenged with a combination of virulent CPV-2b and CPV-2c 5 weeks after vaccination. All puppies in the CPV-2 and CPV-2b vaccinated groups were protected from disease, whereas all control group puppies developed disease and 50% died or were euthanized. This study demonstrated that the CPV-2 and CPV-2b vaccine components of the Continuum DAP and Galaxy DA2PPv products, respectively, provided protection against the CPV-2b virus and also provided complete protection against the new CPV-2c variant.

First isolation of a novel thermostable antifungal peptide secreted by Aspergillus clavatus.

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Skouri-Gargouri, Houda; Gargouri, Ali

2008-11-01

A novel antifungal peptide produced by an indigenous fungal strain (VR) of Aspergillus clavatus was purified. The antifungal peptide was enriched in the supernatant after heat treatment at 70 degrees C. The thermostable character was exploited in the first purification step, as purified peptide was obtained after ultrafiltration and reverse phase-HPLC on C18 column application. The purified peptide named "AcAFP" for A. clavatus antifungal peptide, has molecular mass of 5773Da determined by MALDI-ToF spectrometry. The N-terminal sequence showed a notable identity to the limited family of antifungal peptides produced by ascomycetes fungi. The AcAFP activity remains intact even after heat treatment at 100 degrees C for 1h confirming its thermostability. It exhibits a strong inhibitory activity against mycelial growth of several serious human and plant pathogenic fungi: Fusariuym oxysporum, Fusarium solani, Aspergillus niger, Botrytis cinerea, Alternaria solani, whereas AcAFP did not affect yeast and bacterial growth.

Neutralization antibody response to booster/priming immunization with new equine influenza vaccine in Japan.

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Yamanaka, Takashi; Nemoto, Manabu; Bannai, Hiroshi; Tsujimura, Koji; Matsumura, Tomio; Kokado, Hiroshi; Gildea, Sarah; Cullinane, Ann

2018-03-02

Equine influenza (EI) vaccine has been widely used. However, the causative EI virus (H3N8) undergoes continuous antigenic drift, and the vaccine strains must be periodically reviewed and if necessary, updated to maintain vaccine efficacy against circulating viruses. In 2016, the Japanese vaccine was updated by replacing the old viruses with the Florida sub-lineage Clade (Fc) 2 virus, A/equine/Yokohama/aq13/2010 (Y10). We investigated the virus neutralization (VN) antibody response to Fc2 viruses currently circulating in Europe, after booster or primary immunization with the new vaccine. These European viruses have the amino acid substitution A144V or I179V of the hemagglutinin. In horses that had previously received a primary course and bi-annual boosters with the old vaccine booster, immunization with the updated vaccine increased the VN antibody levels against the European Fc2 viruses as well as Y10. There were no significant differences in the VN titers against Y10 and the Fc2 viruses with A144V or I179V substitution in horses that had received a primary course of the updated vaccine. However, a mixed primary course where the first dose was the old vaccine and the second dose was the updated vaccine, reduced VN titers against the European viruses compared to that against Y10. In summary, the new vaccine affords horses protective level of VN titers against the Fc2 viruses carrying A144V or I179V substitution, but our results suggest that the combination of the old and new vaccines for primary immunization would not be optimum.

Awareness and knowledge about human papillomavirus vaccination and its acceptance in China: a meta-analysis of 58 observational studies

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Yanru Zhang

2016-03-01

Full Text Available Abstract Background The human papillomavirus (HPV vaccines have been widely introduced in immunization programs worldwide, however, it is not accepted in mainland China. We aimed to investigate the awareness and knowledge about HPV vaccines and explore the acceptability of vaccination among the Chinese population. Methods A meta-analysis was conducted across two English (PubMed, EMBASE and three Chinese (China National Knowledge Infrastructure, Wan Fang Database and VIP Database for Chinese Technical Periodicals electronic databases in order to identify HPV vaccination studies conducted in mainland China. We conducted and reported the analysis in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA guidelines. Results Fifty-eight unique studies representing 19 provinces and municipalities in mainland China were assessed. The pooled awareness and knowledge rates about HPV vaccination were 15.95 % (95 % CI: 12.87–19.29, I 2  = 98.9 % and 17.55 % (95 % CI: 12.38–24.88, I 2  = 99.8 %, respectively. The female population (17.39 %; 95 % CI: 13.06–22.20, I 2 = 98.8 % and mixed population (18.55 %; 95 % CI: 14.14–23.42, I 2 = 98.8 % exhibited higher HPV vaccine awareness than the male population (1.82 %; 95 % CI: 0.50–11.20, I 2 = 98.5 %. Populations of mixed ethnicity had lower HPV vaccine awareness (9.61 %; 95 % CI: 5.95–14.03, I 2 = 99.0 % than the Han population (20.17 %; 95 % CI: 16.42–24.20, I 2 = 98.3 %. Among different regions, the HPV vaccine awareness was higher in EDA (17.57 %; 95 % CI: 13.36–22.21, I 2 = 98.0 % and CLDA (17.78 %; 95 % CI: 12.18–24.19, I 2 = 97.6 % than in WUDA (1.80 %; 95 % CI: 0.02–6.33, I 2 = 98.9 %. Furthermore, 67.25 % (95 % CI: 58.75–75.21, I 2  = 99.8 % of participants were willing to be vaccinated, while this number was lower for their daughters (60.32 %; 95 % CI: 51.25–69.04, I 2 �

Brucella suis strain 2 vaccine is safe and protective against heterologous Brucella spp. infections.

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Zhu, Liangquan; Feng, Yu; Zhang, Ge; Jiang, Hui; Zhang, Zhen; Wang, Nan; Ding, Jiabo; Suo, Xun

2016-01-12

Brucellosis is a wide spread zoonotic disease that causes abortion and infertility in mammals and leads to debilitating, febrile illness in humans. Brucella abortus, Brucella melitensis and Brucella suis are the major pathogenic species to humans. Vaccination with live attenuated B. suis strain 2 (S2) vaccine is an essential and critical component in the control of brucellosis in China. The S2 vaccine is very effective in preventing brucellosis in goats, sheep, cattle and swine. However, there are still debates outside of China whether the S2 vaccine is able to provide protection against heterologous virulent Brucella species. We investigated the residual virulence, immunogenicity and protective efficacy of the S2 vaccine in BALB/c mice by determining bacteria persistence in spleen, serum antibody response, cellular immune response and protection against a heterologous virulent challenge. The S2 vaccine was of low virulence as there were no bacteria recovered in spleen four weeks post vaccination. The vaccinated mice developed Brucella-specific IgG in 2-3 weeks, and a burst production of IFN-γ at one week as well as a two-fold increase in TNF-α production. The S2 vaccine protected mice from a virulent challenge by B. melitensis M28, B. abortus 2308 and B. suis S1330, and the S2 vaccinated mice did not develop any clinical signs or tissue damage. Our study demonstrated that the S2 vaccine is of low virulence, stimulates good humoral and cellular immunity and protects animals against infection by heterologous, virulent Brucella species. Copyright © 2015 Elsevier Ltd. All rights reserved.

[Strategies to improve influenza vaccination coverage in Primary Health Care].

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Antón, F; Richart, M J; Serrano, S; Martínez, A M; Pruteanu, D F

2016-04-01

Vaccination coverage reached in adults is insufficient, and there is a real need for new strategies. To compare strategies for improving influenza vaccination coverage in persons older than 64 years. New strategies were introduced in our health care centre during 2013-2014 influenza vaccination campaign, which included vaccinating patients in homes for the aged as well as in the health care centre. A comparison was made on vaccination coverage over the last 4 years in 3 practices of our health care centre: P1, the general physician vaccinated patients older than 64 that came to the practice; P2, the general physician systematically insisted in vaccination in elderly patients, strongly advising to book appointments, and P3, the general physician did not insist. These practices looked after P1: 278; P2: 320; P3: 294 patients older than 64 years. Overall/P1/P2/P3 coverages in 2010: 51.2/51.4/55/46.9% (P=NS), in 2011: 52.4/52.9/53.8/50.3% (P=NS), in 2012: 51.9/52.5/55.3/47.6% (P=NS), and in 2013: 63.5/79.1/59.7/52.7 (P=.000, P1 versus P2 and P3; P=NS between P2 and P3). Comparing the coverages in 2012-2013 within each practice P1 (P=.000); P2 (P=.045); P3 (P=.018). In P2 and P3 all vaccinations were given by the nurses as previously scheduled. In P3, 55% of the vaccinations were given by the nurses, 24.1% by the GP, 9.7% rejected vaccination, and the remainder did not come to the practice during the vaccination period (October 2013-February 2014). The strategy of vaccinating in the homes for the aged improved the vaccination coverage by 5% in each practice. The strategy of "I've got you here, I jab you here" in P1 improved the vaccination coverage by 22%. Copyright © 2014 Sociedad Española de Médicos de Atención Primaria (SEMERGEN). Publicado por Elsevier España, S.L.U. All rights reserved.

Induction of Type I Interferons by Therapeutic Nanoparticle-Based Vaccination Is Indispensable to Reinforce Cytotoxic CD8+ T Cell Responses During Chronic Retroviral Infection

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Torben Knuschke

2018-04-01

Full Text Available T cell dysfunction and immunosuppression are characteristic for chronic viral infections and contribute to viral persistence. Overcoming these burdens is the goal of new therapeutic strategies to cure chronic infectious diseases. We recently described that therapeutic vaccination of chronic retrovirus infected mice with a calcium phosphate (CaP nanoparticle (NP-based vaccine carrier, functionalized with CpG and viral peptides is able to efficiently reactivate the CD8+ T cell response and improve the eradication of virus infected cells. However, the mechanisms underlying this effect were largely unclear. While type I interferons (IFNs I are considered to drive T cell exhaustion by persistent immune activation during chronic viral infection, we here describe an indispensable role of IFN I induced by therapeutic vaccination to efficiently reinforce cytotoxic CD8+ T cells (CTL and improve control of chronic retroviral infection. The induction of IFN I is CpG dependent and leads to significant IFN signaling indicated by upregulation of IFN stimulated genes. By vaccinating chronically retrovirus-infected mice lacking the IFN I receptor (IFNAR−/− or by blocking IFN I signaling in vivo during therapeutic vaccination, we demonstrate that IFN I signaling is necessary to drive full reactivation of CTLs. Surprisingly, we also identified an impaired suppressive capability of regulatory T cells in the presence of IFNα, which implicates an important role for vaccine-induced IFNα in the regulation of the T cell response during chronic retroviral infection. Our data suggest that inducing IFN I signaling in conjunction with the presentation of viral antigens can reactivate immune functions and reduce viral loads in chronic infections. Therefore, we propose CaP NPs as potential therapeutic tool to treat chronic infections.

Human Milk Oligosaccharide 2′-Fucosyllactose Improves Innate and Adaptive Immunity in an Influenza-Specific Murine Vaccination Model

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Ling Xiao

2018-03-01

Full Text Available BackgroundHuman milk is uniquely suited to provide optimal nutrition and immune protection to infants. Human milk oligosaccharides are structural complex and diverse consisting of short chain and long chain oligosaccharides typically present in a 9:1 ratio. 2′-Fucosyllactose (2′FL is one of the most prominent short chain oligosaccharides and is associated with anti-infective capacity of human milk.AimTo determine the effect of 2′FL on vaccination responsiveness (both innate and adaptive in a murine influenza vaccination model and elucidate mechanisms involved.MethodsA dose range of 0.25–5% (w/w dietary 2′FL was provided to 6-week-old female C57Bl/6JOlaHsd mice 2 weeks prior primary and booster vaccination until the end of the experiment. Intradermal (i.d. challenge was performed to measure the vaccine-specific delayed-type hypersensitivity (DTH. Antigen-specific antibody levels in serum as well as immune cell populations within several organs were evaluated using ELISA and flow cytometry, respectively. In an ex vivo restimulation assay, spleen cells were cocultured with influenza-loaded bone marrow-derived dendritic cells (BMDCs to study the effects of 2′FL on vaccine-specific CD4+ and CD8+ T-cell proliferation and cytokine secretions. Furthermore, the direct immune regulatory effects of 2′FL were confirmed using in vitro BMDCs T-cell cocultures.ResultsDietary 2′FL significantly (p < 0.05 enhanced vaccine specific DTH responses accompanied by increased serum levels of vaccine-specific immunoglobulin (Ig G1 and IgG2a in a dose-dependent manner. Consistently, increased activation marker (CD27 expression on splenic B-cells was detected in mice receiving 2′FL as compared to control mice. Moreover, proliferation of vaccine-specific CD4+ and CD8+ T-cells, as well as interferon-γ production after ex vivo restimulation were significantly increased in spleen cells of mice receiving 2′FL as compared to control mice, which were

Multivalent human papillomavirus l1 DNA vaccination utilizing electroporation.

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Kihyuck Kwak

Full Text Available Naked DNA vaccines can be manufactured simply and are stable at ambient temperature, but require improved delivery technologies to boost immunogenicity. Here we explore in vivo electroporation for multivalent codon-optimized human papillomavirus (HPV L1 and L2 DNA vaccination.Balb/c mice were vaccinated three times at two week intervals with a fusion protein comprising L2 residues ∼11-88 of 8 different HPV types (11-88×8 or its DNA expression vector, DNA constructs expressing L1 only or L1+L2 of a single HPV type, or as a mixture of several high-risk HPV types and administered utilizing electroporation, i.m. injection or gene gun. Serum was collected two weeks and 3 months after the last vaccination. Sera from immunized mice were tested for in-vitro neutralization titer, and protective efficacy upon passive transfer to naive mice and vaginal HPV challenge. Heterotypic interactions between L1 proteins of HPV6, HPV16 and HPV18 in 293TT cells were tested by co-precipitation using type-specific monoclonal antibodies.Electroporation with L2 multimer DNA did not elicit detectable antibody titer, whereas DNA expressing L1 or L1+L2 induced L1-specific, type-restricted neutralizing antibodies, with titers approaching those induced by Gardasil. Co-expression of L2 neither augmented L1-specific responses nor induced L2-specific antibodies. Delivery of HPV L1 DNA via in vivo electroporation produces a stronger antibody response compared to i.m. injection or i.d. ballistic delivery via gene gun. Reduced neutralizing antibody titers were observed for certain types when vaccinating with a mixture of L1 (or L1+L2 vectors of multiple HPV types, likely resulting from heterotypic L1 interactions observed in co-immunoprecipitation studies. High titers were restored by vaccinating with individual constructs at different sites, or partially recovered by co-expression of L2, such that durable protective antibody titers were achieved for each type

A new single-dose bivalent vaccine of porcine circovirus type 2 and Mycoplasma hyopneumoniae elicits protective immunity and improves growth performance under field conditions.

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Jeong, Jiwoon; Park, Changhoon; Choi, Kyuhyung; Chae, Chanhee

2016-01-15

The efficacy of the new single-dose bivalent vaccine of porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae was evaluated under field conditions for registration as recommended by the Republic of Korea's Animal, Plant & Fisheries Quarantine & Inspection Agency. Three farms were selected based on their history of co-infection with PCV2 and M. hyopneumoniae. On each farm, a total of 80 3-week-old pigs were randomly allocated to one of two treatment groups: (i) vaccinated (n=40) and (ii) unvaccinated (n=40) animals at 3 weeks of age. Protection by the bivalent vaccine helped increase the market weight by 6.2 kg/pig (106.2 kg in vaccinated group vs. 100 kg in unvaccinated group; Phyopneumoniae-specific IFN-γ-SC. Vaccinated animals displayed a reduced PCV2 load in the blood and M. hyopneumoniae load in nasal swabs compared to unvaccinated animals. Vaccination of pigs against PCV2 and M. hyopneumoniae effectively reduced the lung and lymphoid lesion scores compared to unvaccinated animals in all 3 farms. The new bivalent vaccine is very efficacious in controlling PCV2 and M. hyopneumoniae infection based on clinical, immunological, virological, and pathological evaluations under field conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

Reduction of porcine circovirus type 2 (PCV2 viremia by a reformulated inactivated chimeric PCV1-2 vaccine-induced humoral and cellular immunity after experimental PCV2 challenge

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Seo Hwi

2012-10-01

Full Text Available Abstract Background The objective of the present study was to elucidate the humoral and cellular immune response mechanisms by which a reformulated inactivated chimeric PCV1-2 vaccine reduces the PCV2 viremia. Forty PCV2 seronegative 3-week-old pigs were randomly divided into the following four groups: vaccinated challenged (T01, vaccinated non-challenged (T02, non-vaccinated challenged (T03, and non-vaccinated non-challenged (T04 animals. The pigs in groups T01 and T02 were immunized with a reformulated inactivated chimeric PCV1-2 vaccine (Fostera™ PCV; Pfizer Animal Health administered as a 2.0 ml dose at 21 days of age. At 35 days of age (0 days post-challenge, the pigs in groups T01 and T03 were inoculated intranasally with 2 ml each of PCV2b. Results A reduction of PCV2 viremia coincided with the appearance of both PCV2-specific neutralizing antibodies (NA and interferon-γ-secreting cells (IFN-γ-SCs in the vaccinated animals. However, the presence of anti-PCV2 IgG antibodies did not correlate with the reduction of PCV2 viremia. Lymphocyte subset analysis indicated that the numbers of CD3+ and CD4+ cells increased in vaccinated animals but the numbers of CD4+ cells decreased transiently in non-vaccinated animals. The observation of a delayed type hypersensitivity response in only the vaccinated animals also supports a CD4+ cell-associated protective cellular immune response induced by the reformulated inactivated chimeric PCV1-2 vaccine. Conclusions The induction of PCV2-specific NA and IFN-γ-SCs, and CD4+ cells by the reformulated inactivated chimeric PCV1-2 vaccine is the important protective immune response leading to reduction of the PCV2 viremia and control of the PCV2 infection. To our knowledge this is the first demonstration of protective humoral and cellular immunity induced by the reformulated inactivated chimeric PCV1-2 vaccine and its effect on reduction of PCV2 viremia by vaccination.

Incompatibility of lyophilized inactivated polio vaccine with liquid pentavalent whole-cell-pertussis-containing vaccine

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Kraan, H.; Have, Ten R.; Maas, van der L.; Kersten, G.F.A.; Amorij, J.P.

2016-01-01

A hexavalent vaccine containing diphtheria toxoid, tetanus toxoid, whole cell pertussis, Haemophilius influenza type B, hepatitis B and inactivated polio vaccine (IPV) may: (i) increase the efficiency of vaccination campaigns, (ii) reduce the number of injections thereby reducing needlestick

Safety of Quadrivalent Meningococcal Conjugate Vaccine in Children 2-10 Years.

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Tartof, Sara Yee; Sy, Lina S; Ackerson, Bradley K; Hechter, Rulin C; Haag, Mendel; Slezak, Jeffrey M; Luo, Yi; Fischetti, Christine A; Takhar, Harp S; Miao, Yan; Solano, Zendi; Jacobsen, Steven J; Tseng, Hung-Fu

2017-11-01

Quadrivalent meningococcal conjugate vaccine is recommended for children, adolescents and adults at increased risk of meningococcal disease. In 2011, MenACWY-CRM (Menveo, GSK, Siena, Italy) was approved for children 2-10 years of age in the United States. Although no safety concerns arose from clinical trials, it remains important to monitor its safety in routine clinical settings. Kaiser Permanente Southern California members 2-10 years old who received MenACWY-CRM between September 2011 and September 2014 were included. Electronic health records were searched using a validated algorithm to identify 26 prespecified events of interests (EOIs) and serious medically attended events (SMAEs) from inpatient or emergency settings up to 1 year after MenACWY-CRM vaccination. SMAEs were categorized by International Classification of Diseases, 9th revision diagnostic categories. All events were reviewed to confirm the diagnosis and symptom onset date. The study was descriptive (NCT01452438); no statistical tests were performed. Among 387 vaccinated children, 327 with ≥6 months membership before vaccination were analyzed. Among EOIs, 9 asthma cases and 1 myasthenia gravis case underwent chart review which confirmed 1 incident asthma case occurring 237 days after concomitant vaccination with MenACWY-CRM and typhoid vaccine. Thirty-one children experienced SMAEs, most commonly because of unrelated injury and poisoning. The remaining events occurred sporadically after vaccination and most were unlikely related to vaccination based on medical record review. One incident EOI of asthma late in the 1-year observation period and sporadic distribution of SMAEs were observed. These data do not suggest safety concerns associated with MenACWY-CRM vaccination in children 2-10 years old.

Phase I vaccination trial of SYT-SSX junction peptide in patients with disseminated synovial sarcoma

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Asanuma Hiroko

2005-01-01

Full Text Available Abstract Background Synovial sarcoma is a high-grade malignant tumor of soft tissue, characterized by the specific chromosomal translocation t(X;18, and its resultant SYT-SSX fusion gene. Despite intensive multimodality therapy, the majority of metastatic or relapsed diseases still remain incurable, thus suggesting a need for new therapeutic options. We previously demonstrated the antigenicity of SYT-SSX gene-derived peptides by in vitro analyses. The present study was designed to evaluate in vivo immunological property of a SYT-SSX junction peptide in selected patients with synovial sarcoma. Methods A 9-mer peptide (SYT-SSX B: GYDQIMPKK spanning the SYT-SSX fusion region was synthesized. Eligible patients were those (i who have histologically and genetically confirmed, unresectable synovial sarcoma (SYT-SSX1 or SYT-SSX2 positive, (ii HLA-A*2402 positive, (iii between 20 and 70 years old, (iv ECOG performance status between 0 and 3, and (v who gave informed consent. Vaccinations with SYT-SSX B peptide (0.1 mg or 1.0 mg were given subcutaneously six times at 14-day intervals. These patients were evaluated for DTH skin test, adverse events, tumor size, tetramer staining, and peptide-specific CTL induction. Results A total of 16 vaccinations were carried out in six patients. The results were (i no serious adverse effects or DTH reactions, (ii suppression of tumor progression in one patient, (iii increases in the frequency of peptide-specific CTLs in three patients and a decrease in one patient, and (iv successful induction of peptide-specific CTLs from four patients. Conclusions Our findings indicate the safety of the SYT-SSX junction peptide in the use of vaccination and also give support to the property of the peptide to evoke in vivo immunological responses. Modification of both the peptide itself and the related protocol is required to further improve the therapeutic efficacy.

Effect of zymosan and poly (I:C) adjuvants on responses to microneedle immunization coated with whole inactivated influenza vaccine.

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Shin, Ju-Hyung; Noh, Jin-Yong; Kim, Kwon-Ho; Park, Jae-Keun; Lee, Ji-Ho; Jeong, Seong Dong; Jung, Dae-Yoon; Song, Chang-Seon; Kim, Yeu-Chun

2017-11-10

Microneedles are the micrometer size devices used for the delivery of vaccines and biotherapeutics. In order to increase the vaccine efficacy and reduce the antigen dose, there is a significant need to find some adjuvants for the microneedle vaccination. In this study, zymosan, which is the cell wall preparation of Saccharomyces cerevisiae, or poly (I:C) was coated on a microneedle with inactivated influenza virus, and then immunized into BALB/c mouse to determine the immunogenicity, protection and synergetic effect between two adjuvants. As a result, the group administered with zymosan and vaccine antigen showed significantly stronger IgG response, HI titer and IgG subtypes without any adverse effects, compared to the group immunized with the vaccine antigen alone. Also, there were enhanced cellular immune responses in the group received adjuvant with vaccine antigen. In addition, they showed superior protection and lung viral reduction against lethal viral challenge. Taken together, this study confirms that zymosan can be used as an immunostimulant for microneedle vaccination. Copyright © 2017 Elsevier B.V. All rights reserved.

Next generation sequencing of DNA-launched Chikungunya vaccine virus

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Hidajat, Rachmat; Nickols, Brian [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Forrester, Naomi [Institute for Human Infections and Immunity, Sealy Center for Vaccine Development and Department of Pathology, University of Texas Medical Branch, GNL, 301 University Blvd., Galveston, TX 77555 (United States); Tretyakova, Irina [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Weaver, Scott [Institute for Human Infections and Immunity, Sealy Center for Vaccine Development and Department of Pathology, University of Texas Medical Branch, GNL, 301 University Blvd., Galveston, TX 77555 (United States); Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States)

2016-03-15

Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3′ untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at the E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety. - Highlights: • Chikungunya virus (CHIKV) is an emerging pandemic threat. • In vivo DNA-launched attenuated CHIKV is a novel vaccine technology. • DNA-launched virus was sequenced using HiSeq2000 and compared to the 181/25 virus. • DNA-launched virus has lower frequency of SNPs at E2-12 and E2-82 attenuation loci.

Next generation sequencing of DNA-launched Chikungunya vaccine virus

International Nuclear Information System (INIS)

Hidajat, Rachmat; Nickols, Brian; Forrester, Naomi; Tretyakova, Irina; Weaver, Scott; Pushko, Peter

2016-01-01

Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3′ untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at the E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety. - Highlights: • Chikungunya virus (CHIKV) is an emerging pandemic threat. • In vivo DNA-launched attenuated CHIKV is a novel vaccine technology. • DNA-launched virus was sequenced using HiSeq2000 and compared to the 181/25 virus. • DNA-launched virus has lower frequency of SNPs at E2-12 and E2-82 attenuation loci.

Recovery of avirulent, thermostable Newcastle disease virus strain NDV4-C from cloned cDNA and stable expression of an inserted foreign gene

NARCIS (Netherlands)

Zhang, X.; Liu, H.; Liu, P.; Peeters, B.P.H.; Zhao, C.; Kong, X.

2013-01-01

A reverse genetics system for thermostable Newcastle disease virus (NDV) is not currently available. In this study, we developed a reverse genetics system for the avirulent and thermostable NDV4-C strain. Successful recovery of NDV4-C was achieved by using either T7 RNA polymerase or cellular RNA

Immunogenicity and safety of a CRM-conjugated meningococcal ACWY vaccine administered concomitantly with routine vaccines starting at 2 months of age.

Science.gov (United States)

Nolan, Terry M; Nissen, Michael D; Naz, Aftab; Shepard, Julie; Bedell, Lisa; Hohenboken, Matthew; Odrljin, Tatjana; Dull, Peter M

2014-01-01

Infants are at the highest risk for meningococcal disease and a broadly protective and safe vaccine is an unmet need in this youngest population. We evaluated the immunogenicity and safety of a 4-dose infant/toddler regimen of MenACWY-CRM given at 2, 4, 6, and 12 months of age concomitantly with pentavalent diphtheria-tetanus-acellular pertussis-Hemophilus influenzae type b-inactivated poliovirus-combination vaccine (DTaP-IPV/Hib), hepatitis B vaccine (HBV), 7- or 13-valent conjugate pneumococcal vaccine (PCV), and measles, mumps, and rubella vaccine (MMR). Four doses of MenACWY-CRM induced hSBA titers ≥8 in 89%, 95%, 97%, and 96% of participants against serogroups A, C, W-135, and Y, respectively. hSBA titers ≥8 were present in 76-98% of participants after the first 3 doses. A categorical linear analysis incorporating vaccine group and study center showed responses to routine vaccines administered with MenACWY-CRM were non-inferior to routine vaccines alone, except for seroresponse to the pertussis antigen fimbriae. The reactogenicity profile was not affected when MenACWY-CRM was administered concomitantly with routine vaccines. MenACWY-CRM administered with routine concomitant vaccinations in young infants was well tolerated and induced highly immunogenic responses against each of the serogroups without significant interference with the immune responses to routine infant vaccinations. Healthy 2 month old infants were randomized to receive MenACWY-CRM with routine vaccines (n = 258) or routine vaccines alone (n = 271). Immunogenicity was assessed by serum bactericidal assay using human complement (hSBA). Medically attended adverse events (AEs), serious AEs (SAEs) and AEs leading to study withdrawal were collected throughout the study period.

The ''THERMOST'' for analysing thermo-structural behaviour of LWR fuel rod under PCI conditions

International Nuclear Information System (INIS)

Nuno, H.; Ogawa, S.; Kobayashi, H.

1983-01-01

As one of the methods for evaluating the fuel rod performances under power ramping or load following operations, the combined ''FROST'' and ''THERMOST'' system has been developed and being brought into practical use. The former had already been presented at Blackpool Meeting in 1978, and the latter is going to be presented in this paper. The major purpose of the THERMOST is to analyse very detailed thermal and structural fuel behaviours in a rather localized part of fuel rod whereas the FROST deals with whole-rod-wide general performances. The code handles 2-dimensional thermal and structural analyses simultaneously by using finite element method, in axial section wide or in lateral section wide. It consists of a fundamental FEM system of generalized constitution and its surrounding subroutine system which characterizes fuel behaviours such as temperature distribution, thermal expansion, elastoplasticity, creep, cracking, swelling, growth, etc. Thermal analysis is handled by heat conduction and heat transfer elements (6 kinds) and structural analysis by axisymmetric ring and lateral plane elements (6 kinds). Boundary problems such as contact, friction and cracking are treated by gap and crack elements. A sample calculation of PCI performance on a PWR fuel rod under ramping condition is presented with some inpile test data. (author)

Phosphate and arsenate removal efficiency by thermostable ferritin enzyme from Pyrococcus furiosus using radioisotopes

KAUST Repository

Sevcenco, Ana-Maria; Paravidino, Monica; Vrouwenvelder, Johannes S.; Wolterbeek, Hubert Th.; van Loosdrecht, Mark C.M.; Hagen, Wilfred R.

2015-01-01

Oxo-anion binding properties of the thermostable enzyme ferritin from Pyrococcus furiosus were characterized with radiography. Radioisotopes 32P and 76As present as oxoanions were used to measure the extent and the rate of their absorption

Enzymatic liquefaction of agarose above the sol-gel transition temperature using a thermostable endo-type β-agarase, Aga16B.

Science.gov (United States)

Kim, Jung Hyun; Yun, Eun Ju; Seo, Nari; Yu, Sora; Kim, Dong Hyun; Cho, Kyung Mun; An, Hyun Joo; Kim, Jae-Han; Choi, In-Geol; Kim, Kyoung Heon

2017-02-01

The main carbohydrate of red macroalgae is agarose, a heterogeneous polysaccharide composed of D-galactose and 3,6-anhydro-L-galactose. When saccharifying agarose by enzymes, the unique physical properties of agarose, namely the sol-gel transition and the near-insolubility of agarose in water, limit the accessibility of agarose to the enzymes. Due to the lower accessibility of agarose to enzymes in the gel state than to the sol state, it is important to prevent the sol-gel transition by performing the enzymatic liquefaction of agarose at a temperature higher than the sol-gel transition temperature of agarose. In this study, a thermostable endo-type β-agarase, Aga16B, originating from Saccharophagus degradans 2-40 T , was characterized and introduced in the liquefaction process. Aga16B was thermostable up to 50 °C and depolymerized agarose mainly into neoagarooligosaccharides with degrees of polymerization 4 and 6. Aga16B was applied to enzymatic liquefaction of agarose at 45 °C, which was above the sol-gel transition temperature of 1 % (w/v) agarose (∼35 °C) when cooling agarose. This is the first systematic demonstration of enzymatic liquefaction of agarose, enabled by determining the sol-gel temperature of agarose under specific conditions and by characterizing the thermostability of an endo-type β-agarase.

Not so monofunctional-a case of thermostable Thermobifida fusca catalase with peroxidase activity

NARCIS (Netherlands)

Lončar, Nikola; Fraaije, Marco W

Thermobifida fusca is a mesothermophilic organism known for its ability to degrade plant biomass and other organics, and it was demonstrated that it represents a rich resource of genes encoding for potent enzymes for biocatalysis. The thermostable catalase from T. fusca has been cloned and

Optimization of multimeric human papillomavirus L2 vaccines.

Directory of Open Access Journals (Sweden)

Subhashini Jagu

Full Text Available We sought to define the protective epitopes within the amino terminus of human papillomavirus (HPV type 16 minor capsid protein L2. Passive transfer of mice with rabbit antisera to HPV16 L2 peptides 17-36, 32-51 and 65-81 provided significant protection against vaginal HPV16 challenge, whereas antisera to 47-66, 108-120 or 373-392 did not. Vaccination with L1 virus-like particles induces a high titer, but generally type-restricted neutralizing antibody response. Conversely, vaccination with L2 11-88, especially multimers thereof, induces antibodies that neutralize a broad range of papillomavirus types, albeit at lower titers than for L1 VLP. With the intent of enhancing the immunogenicity and the breadth of protection by focusing the immune response to the key protective epitopes, we designed L2 fusion proteins consisting of residues ∼11-88 of eight divergent mucosal HPV types 6, 16, 18, 31, 39, 51, 56, 73 (11-88×8 or residues ∼13-47 of fifteen HPV types (13-47×15. The 11-88×8 was significantly more immunogenic than 13-47×15 in Balb/c mice regardless of the adjuvant used, suggesting the value of including the 65-81 protective epitope in the vaccine. Since the L2 47-66 peptide antiserum failed to elicit significant protection, we generated an 11-88×8 construct deleted for this region in each subunit (11-88×8Δ. Mice were vaccinated with 11-88×8 and 11-88×8Δ to determine if deletion of this non-protective epitope enhanced the neutralizing antibody response. However, 11-88×8Δ was significantly less immunogenic than 11-88×8, and even the addition of a known T helper epitope, PADRE, to the construct (11-88×8ΔPADRE failed to recover the immunogenicity of 11-88×8 in C57BL/6 mice, suggesting that while L2 47-66 is not a critical protective or T helper epitope, it nevertheless contributes to the immunogenicity of the L2 11-88×8 multimer vaccine.

Vaccination against Canine Distemper Virus Infection in Infant Ferrets with and without Maternal Antibody Protection, Using Recombinant Attenuated Poxvirus Vaccines

Science.gov (United States)

Welter, Janet; Taylor, Jill; Tartaglia, James; Paoletti, Enzo; Stephensen, Charles B.

2000-01-01

Canine distemper virus (CDV) infection of ferrets is clinically and immunologically similar to measles, making this a useful model for the human disease. The model was used to determine if parenteral or mucosal immunization of infant ferrets at 3 and 6 weeks of age with attenuated vaccinia virus (NYVAC) or canarypox virus (ALVAC) vaccine strains expressing the CDV hemagglutinin (H) and fusion (F) protein genes (NYVAC-HF and ALVAC-HF) would induce serum neutralizing antibody and protect against challenge infection at 12 weeks of age. Ferrets without maternal antibody that were vaccinated parenterally with NYVAC-HF (n = 5) or ALVAC-HF (n = 4) developed significant neutralizing titers (log10 inverse mean titer ± standard deviation of 2.30 ± 0.12 and 2.20 ± 0.34, respectively) by the day of challenge, and all survived with no clinical or virologic evidence of infection. Ferrets without maternal antibody that were vaccinated intranasally (i.n.) developed lower neutralizing titers, with NYVAC-HF producing higher titers at challenge (1.11 ± 0.57 versus 0.40 ± 0.37, P = 0.02) and a better survival rate (6/7 versus 0/5, P = 0.008) than ALVAC-HF. Ferrets with maternal antibody that were vaccinated parenterally with NYVAC-HF (n = 7) and ALVAC-HF (n = 7) developed significantly higher antibody titers (1.64 ± 0.54 and 1.28 ± 0.40, respectively) than did ferrets immunized with an attenuated CDV vaccine (0.46 ± 0.59; n = 7) or the recombinant vectors expressing rabies glycoprotein (RG) (0.19 ± 0.32; n = 8, P = 7 × 10−6). The NYVAC vaccine also protected against weight loss, and both the NYVAC and attenuated CDV vaccines protected against the development of some clinical signs of infection, although survival in each of the three vaccine groups was low (one of seven) and not significantly different from the RG controls (none of eight). Combined i.n.-parenteral immunization of ferrets with maternal antibody using NYVAC-HF (n = 9) produced higher titers (1.63 ± 0

Vaccination against canine distemper virus infection in infant ferrets with and without maternal antibody protection, using recombinant attenuated poxvirus vaccines.

Science.gov (United States)

Welter, J; Taylor, J; Tartaglia, J; Paoletti, E; Stephensen, C B

2000-07-01

Canine distemper virus (CDV) infection of ferrets is clinically and immunologically similar to measles, making this a useful model for the human disease. The model was used to determine if parenteral or mucosal immunization of infant ferrets at 3 and 6 weeks of age with attenuated vaccinia virus (NYVAC) or canarypox virus (ALVAC) vaccine strains expressing the CDV hemagglutinin (H) and fusion (F) protein genes (NYVAC-HF and ALVAC-HF) would induce serum neutralizing antibody and protect against challenge infection at 12 weeks of age. Ferrets without maternal antibody that were vaccinated parenterally with NYVAC-HF (n = 5) or ALVAC-HF (n = 4) developed significant neutralizing titers (log(10) inverse mean titer +/- standard deviation of 2.30 +/- 0.12 and 2.20 +/- 0.34, respectively) by the day of challenge, and all survived with no clinical or virologic evidence of infection. Ferrets without maternal antibody that were vaccinated intranasally (i.n.) developed lower neutralizing titers, with NYVAC-HF producing higher titers at challenge (1.11 +/- 0.57 versus 0.40 +/- 0.37, P = 0.02) and a better survival rate (6/7 versus 0/5, P = 0.008) than ALVAC-HF. Ferrets with maternal antibody that were vaccinated parenterally with NYVAC-HF (n = 7) and ALVAC-HF (n = 7) developed significantly higher antibody titers (1.64 +/- 0. 54 and 1.28 +/- 0.40, respectively) than did ferrets immunized with an attenuated CDV vaccine (0.46 +/- 0.59; n = 7) or the recombinant vectors expressing rabies glycoprotein (RG) (0.19 +/- 0.32; n = 8, P = 7 x 10(-6)). The NYVAC vaccine also protected against weight loss, and both the NYVAC and attenuated CDV vaccines protected against the development of some clinical signs of infection, although survival in each of the three vaccine groups was low (one of seven) and not significantly different from the RG controls (none of eight). Combined i.n.-parenteral immunization of ferrets with maternal antibody using NYVAC-HF (n = 9) produced higher titers (1

Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-β-mannosidase from Aspergillus niger BK01

Directory of Open Access Journals (Sweden)

Sigoillot Jean-Claude

2009-11-01

Full Text Available Abstract Background Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-β-mannosidases (1,4-β-D-mannanases catalyze the random hydrolysis of β-1,4-mannosidic linkages in the main chain of β-mannans. Biodegradation of β-mannans by the action of thermostable mannan endo-1,4-β-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS. Results A gene encoding mannan endo-1,4-β-mannosidase or 1,4-β-D-mannan mannanohydrolase (E.C. 3.2.1.78, commonly termed β-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5, was cloned and successfully expressed heterologously (up to 243 μg of active recombinant protein per mL in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant β-mannanase is highly thermostable with a half-life time of approximately 56 h at 70°C and pH 4.0. The optimal temperature (10-min assay and pH value for activity are 80°C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Km values of the enzyme for konjac glucomannan (low viscosity, locust bean gum galactomannan, carob galactomannan (low viscosity, and 1,4-β-D-mannan (from carob are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1 and 1.5 mg mL-1, respectively, while the kcat values for these

Humoral response to 2 inactivated bluetongue virus serotype-8 vaccines in South American camelids.

Science.gov (United States)

Zanolari, P; Bruckner, L; Fricker, R; Kaufmann, C; Mudry, M; Griot, C; Meylan, M

2010-01-01

Bluetongue virus serotype 8 (BTV-8) has caused disease in domestic ruminants in several countries of northern Europe since 2006. In 2008 a mass-vaccination program was launched in most affected countries using whole virus inactivated vaccines. To evaluate 2 inactivated vaccines (Bovilis BTV 8; BTVPUR AlSap8) for immunogenicity and safety against BTV-8 in South American camelids (SAC) in a field trial. Forty-two SAC (25 Alpacas, 17 Llamas) aged between 1 and 16 years. The animals were vaccinated twice at intervals of 21 days. They were observed clinically for adverse local, systemic, or both reactions throughout the trial. Blood samples collected on days 0, 14, 21, 43, and 156 after vaccination were tested for the presence of BTV-8 virus by real time-polymerase chain reaction and of specific antibodies by competitive ELISA and a serum neutralization test. All vaccinated animals developed antibodies to BTV-8 after the 2nd administration of the vaccine. No adverse effects were observed except for moderate local swellings at the injection site, which disappeared within 21 days. Slightly increased body temperatures were only observed in the first 2 days after vaccination. The BTV was not detected in any of the samples analyzed. The administration of the 2 inactivated commercial vaccines was safe and induced seroconversion against BTV-8 in all vaccinated animals. The results of this study suggest that 2 doses injected 3 weeks apart is a suitable vaccination regimen for SAC.

Compatibility of a bivalent modified-live vaccine against Bordetella bronchiseptica and CPiV, and a trivalent modified-live vaccine against CPV, CDV and CAV-2.

Science.gov (United States)

Jacobs, A A C; Bergman, J G H E; Theelen, R P H; Jaspers, R; Helps, J M; Horspool, L J I; Paul, G

2007-01-13

Eight puppies (group 1) were vaccinated once with a bivalent modified-live vaccine against infectious tracheobronchitis by the intranasal route and at the same time with an injectable trivalent vaccine against canine parvovirus, canine distemper virus and canine adenovirus; a second group of eight puppies (group 2) was vaccinated only with the intranasal bivalent vaccine, and a further eight puppies (group 3) were vaccinated only with the injectable trivalent vaccine. Three weeks later they were all challenged with wildtype Bordetella bronchiseptica and canine parainfluenza virus by the aerosol route, and their antibody responses to the five vaccine organisms were determined. Oronasal swabs were taken regularly before and after the challenge for the isolation of bacteria and viruses, and the puppies were observed for clinical signs for three weeks after the challenge. There were no significant differences in the puppies' titres against canine parvovirus, canine distemper virus and canine adenovirus type 2 between the groups vaccinated with or without the bivalent intranasal vaccine. After the challenge the mean clinical scores of the two groups vaccinated with the intranasal vaccine were nearly 90 per cent lower (P=0.001) than the mean score of the group vaccinated with only the trivalent injectable vaccine, and the puppies in this group all became culture-positive for B bronchiseptica and canine parainfluenza virus. There were only small differences between the rates of isolation of B bronchiseptica from groups 1, 2 and 3, but significantly lower yields of canine parainfluenza virus were isolated from groups 1 and 2 than from group 3.

Use of the mice passive protection test to evaluate the humoral response in goats vaccinated with Sterne 34F2 live spore vaccine.

Science.gov (United States)

Phaswana, P H; Ndumnego, O C; Koehler, S M; Beyer, W; Crafford, J E; van Heerden, H

2017-09-07

The Sterne live spore vaccine (34F2) is the most widely used veterinary vaccine against anthrax in animals. Antibody responses to several antigens of Bacillus anthracis have been described with a large focus on those against protective antigen (PA). The focus of this study was to evaluate the protective humoral immune response induced by the live spore anthrax vaccine in goats. Boer goats vaccinated twice (week 0 and week 12) with the Sterne live spore vaccine and naive goats were used to monitor the anti-PA and toxin neutralizing antibodies at week 4 and week 17 (after the second vaccine dose) post vaccination. A/J mice were passively immunized with different dilutions of sera from immune and naive goats and then challenged with spores of B. anthracis strain 34F2 to determine the protective capacity of the goat sera. The goat anti-PA ELISA titres indicated significant sero-conversion at week 17 after the second doses of vaccine (p = 0.009). Mice receiving undiluted sera from goats given two doses of vaccine (twice immunized) showed the highest protection (86%) with only 20% of mice receiving 1:1000 diluted sera surviving lethal challenge. The in vitro toxin neutralization assay (TNA) titres correlated to protection of passively immunized A/J mice against lethal infection with the vaccine strain Sterne 34F2 spores using immune goat sera up to a 1:10 dilution (r s  ≥ 0.522, p = 0.046). This study suggests that the passive mouse protection model could be potentially used to evaluate the protective immune response in livestock animals vaccinated with the current live vaccine and new vaccines.

Udvikling af antistoffer efter vaccination mod og podning med PRRSV

DEFF Research Database (Denmark)

Sonne Kristensen, Charlotte; Qvist Pawlowski, Mia; Thoning, Henrik

konkludere, at det er muligt at vaccinere med begge PRRS vacciner på samme tid og opnå antistofsvar for begge typer PRRSV. Husk dog altid at give forskellige vacciner i hver sin side af nakken. Blodprøver fra grisene viste, at de havde dannet antistoffer og blev positive i ELISA-testen for PRRSV Type 1 21......-US samt IPT testene. Disse tests kan dog ikke skelne ”eksotiske” PRRSV subtyper fra de almindelige cirkulerende PRRSV subtyper i Danmark. Forsøget blev gennemført på Lindholm. Der indgik 66 grise fra en Blå SPF+Ap6+Ap12 besætning. Grisene blev ved ankomst fordelt i 4 grupper (VAC-T1, VAC-T2, VAC-T1T2 og...... NON-VAC): VAC-T1 blev vaccineret med Porcilis ® PRRS VET VAC-T2 blev vaccineret med Ingelvac ® PRRS VET VAC-T1T2 fik begge vacciner samtidig i hver sin side af nakken NON-VAC blev ikke vaccineret. Grisene blev gennem hele forsøget observeret dagligt. De fik taget temperatur den første uge efter...

Thermostability of bovine submaxillary mucin (BSM) in bulk solution and at a sliding interface

DEFF Research Database (Denmark)

Madsen, Jan Busk; Pakkanen, Kirsi I.; Lee, Seunghwan

2014-01-01

Thermostability of bovine submaxillary mucin (BSM) was studied in terms of its structure, hydrodynamic size, surface adsorption, and lubricating properties in the temperature range of 5-85°C. The overall random coil structure of BSM showed a gradual loosening with increasing temperature as charac......Thermostability of bovine submaxillary mucin (BSM) was studied in terms of its structure, hydrodynamic size, surface adsorption, and lubricating properties in the temperature range of 5-85°C. The overall random coil structure of BSM showed a gradual loosening with increasing temperature...... as characterized by circular dichroism (CD) spectroscopy, but this change was fully reversible upon lowering temperature. Extended heating up to 120min at 80°C did not make any appreciable changes in the structure of BSM when it was cooled to room temperature. The hydrodynamic size of BSM, as studied by dynamic...

Synthetic TLR4 agonists enhance functional antibodies and CD4+ T-cell responses against the Plasmodium falciparum GMZ2.6C multi-stage vaccine antigen

NARCIS (Netherlands)

Baldwin, S.L.; Roeffen, W.; Singh, S.K; Tiendrebeogo, R.W.; Christiansen, M.; Beebe, E.; Carter, D.; Fox, C.B.; Howard, R.F.; Reed, S.G.; Sauerwein, R.; Theisen, M.

2016-01-01

A subunit vaccine targeting both transmission and pathogenic asexual blood stages of Plasmodium falciparum, i.e., a multi-stage vaccine, could be a powerful tool to combat malaria. Here, we report production and characterization of the recombinant protein GMZ2.6C, which contains a fragment of the

Efficacy and safety of simultaneous vaccination with two modified live virus vaccines against porcine reproductive and respiratory syndrome virus types 1 and 2 in pigs

DEFF Research Database (Denmark)

Kristensen, Charlotte S.; Kvisgaard, Lise Kirstine; Pawlowski, Maciej

2018-01-01

from groups 1–4 were mingled in new groups and challenged (DPC 0) with PRRSV-1, subtype 1, PRRSV-1, subtype 2 or PRRSV-2. On DPC 13/14 all pigs were necropsied. Samples were collected after vaccination and challenge. PRRSV was detected in all vaccinated pigs and the majority of the pigs were positive...... there was limited effect on the viral load in serum following challenge with the PRRSV-1 strains. Vaccination against PRRSV-1 had less impact on viremia following challenge. The protective effects of simultaneous vaccination with PRRSV Type 1 and 2 MLV vaccines and single PRRS MLV vaccination were comparable. None....... Thus, simultaneous administration of the two vaccines is an option in herds with both PRRSV types....

Induction of indoleamine 2, 3-dioxygenase in human dendritic cells by a cholera toxin B subunit-proinsulin vaccine.

Directory of Open Access Journals (Sweden)

Jacques C Mbongue

Full Text Available Dendritic cells (DC interact with naïve T cells to regulate the delicate balance between immunity and tolerance required to maintain immunological homeostasis. In this study, immature human dendritic cells (iDC were inoculated with a chimeric fusion protein vaccine containing the pancreatic β-cell auto-antigen proinsulin linked to a mucosal adjuvant the cholera toxin B subunit (CTB-INS. Proteomic analysis of vaccine inoculated DCs revealed strong up-regulation of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1. Increased biosynthesis of the immunosuppressive enzyme was detected in DCs inoculated with the CTB-INS fusion protein but not in DCs inoculated with proinsulin, CTB, or an unlinked combination of the two proteins. Immunoblot and PCR analyses of vaccine treated DCs detected IDO1mRNA by 3 hours and IDO1 protein synthesis by 6 hours after vaccine inoculation. Determination of IDO1 activity in vaccinated DCs by measurement of tryptophan degradation products (kynurenines showed increased tryptophan cleavage into N-formyl kynurenine. Vaccination did not interfere with monocytes differentiation into DC, suggesting the vaccine can function safely in the human immune system. Treatment of vaccinated DCs with pharmacological NF-κB inhibitors ACHP or DHMEQ significantly inhibited IDO1 biosynthesis, suggesting a role for NF-κB signaling in vaccine up-regulation of dendritic cell IDO1. Heat map analysis of the proteomic data revealed an overall down-regulation of vaccinated DC functions, suggesting vaccine suppression of DC maturation. Together, our experimental data indicate that CTB-INS vaccine induction of IDO1 biosynthesis in human DCs may result in the inhibition of DC maturation generating a durable state of immunological tolerance. Understanding how CTB-INS modulates IDO1 activity in human DCs will facilitate vaccine efficacy and safety, moving this immunosuppressive strategy closer to clinical applications for prevention

Health and economic outcomes of HPV 16,18 vaccination in 72 GAVI-eligible countries.

Science.gov (United States)

Goldie, Sue J; O'Shea, Meredith; Campos, Nicole Gastineau; Diaz, Mireia; Sweet, Steven; Kim, Sun-Young

2008-07-29

The risk of dying from cervical cancer is disproportionately borne by women in developing countries. Two new vaccines are highly effective in preventing HPV 16,18 infection, responsible for approximately 70% of cervical cancer, in girls not previously infected. The GAVI Alliance (GAVI) provides technical assistance and financial support for immunization in the world's poorest countries. Using population-based and epidemiologic data for 72 GAVI-eligible countries we estimate averted cervical cancer cases and deaths, disability-adjusted years of life (DALYs) averted and incremental cost-effectiveness ratios (I$/DALY averted) associated with HPV 16,18 vaccination of young adolescent girls. In addition to vaccine coverage and efficacy, relative and absolute cancer reduction depended on underlying incidence, proportion attributable to HPV types 16 and 18, population age-structure and competing mortality. With 70% coverage, mean reduction in the lifetime risk of cancer is below 40% in some countries (e.g., Nigeria, Ghana) and above 50% in others (e.g., India, Uganda, Kenya). At I$10 per vaccinated girl (approximately $2.00 per dose assuming three doses, plus wastage, administration, program support) vaccination was cost-effective in all countries using a per capita GDP threshold; for 49 of 72 countries, the cost per DALY averted was less than I$100 and for 59 countries, it was less than I$200. Taking into account country-specific assumptions (per capita GNI, DPT3 coverage, percentage of girls who are enrolled in fifth grade) for the year of introduction, percent coverage achieved in the first year, and years to maximum coverage, a 10-year modeled scenario prevented the future deaths of approximately 2 million women vaccinated as adolescents. Despite favorable cost-effectiveness, assessment of financial costs raised concerns about affordability; as the cost per vaccinated girl was increased from I$10 to I$25 (approximately $2 to $5 per dose), the financial costs for the

Influenza vaccine-mediated protection in older adults: Impact of influenza infection, cytomegalovirus serostatus and vaccine dosage.

Science.gov (United States)

Merani, Shahzma; Kuchel, George A; Kleppinger, Alison; McElhaney, Janet E

2018-07-01

Age-related changes in T-cell function are associated with a loss of influenza vaccine efficacy in older adults. Both antibody and cell-mediated immunity plays a prominent role in protecting older adults, particularly against the serious complications of influenza. High dose (HD) influenza vaccines induce higher antibody titers in older adults compared to standard dose (SD) vaccines, yet its impact on T-cell memory is not clear. The aim of this study was to compare the antibody and T-cell responses in older adults randomized to receive HD or SD influenza vaccine as well as determine whether cytomegalovirus (CMV) serostatus affects the response to vaccination, and identify differences in the response to vaccination in those older adults who subsequently have an influenza infection. Older adults (≥65years) were enrolled (n=106) and randomized to receive SD or HD influenza vaccine. Blood was collected pre-vaccination, followed by 4, 10 and 20weeks post-vaccination. Serum antibody titers, as well as levels of inducible granzyme B (iGrB) and cytokines were measured in PBMCs challenged ex vivo with live influenza virus. Surveillance conducted during the influenza season identified those with laboratory confirmed influenza illness or infection. HD influenza vaccination induced a high antibody titer and IL-10 response, and a short-lived increase in Th1 responses (IFN-γ and iGrB) compared to SD vaccination in PBMCs challenged ex vivo with live influenza virus. Of the older adults who became infected with influenza, a high IL-10 and iGrB response in virus-challenged cells was observed post-infection (week 10 to 20), as well as IFN-γ and TNF-α at week 20. Additionally, CMV seropositive older adults had an impaired iGrB response to influenza virus-challenge, regardless of vaccine dose. This study illustrates that HD influenza vaccines have little impact on the development of functional T-cell memory in older adults. Furthermore, poor outcomes of influenza infection in

Crystallization and preliminary X-ray crystallographic analysis of l-rhamnose isomerase with a novel high thermostability from Bacillus halodurans

International Nuclear Information System (INIS)

Doan, Thi-Ngoc-Thanh; Prabhu, Ponnandy; Kim, Jin-Kwang; Ahn, Yeh-Jin; Natarajan, Sampath; Kang, Lin-Woo; Park, Geon Tae; Lim, Sang-Boem; Lee, Jung-Kul

2010-01-01

l-Rhamnose isomerase (l-RhI) from B. halodurans has been purified and crystallized. The crystals of l-RhI belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 83.2, b = 164.9, c = 92.0 Å, β = 116.0°, and diffracted to 2.5 Å resolution. l-Rhamnose isomerases catalyze isomerization between l-rhamnose (6-deoxy-l-mannose) and l-rhamnulose (6-deoxy-l-fructose), which is the first step in rhamnose catabolism. l-Rhamnose isomerase from Bacillus halodurans ATCC BAA-125 (BHRI) exhibits interesting characteristics such as high thermostability and selective substrate specificity. BHRI fused with an HHHHHH sequence was purified and crystallized in order to elucidate the molecular basis of its unique enzymatic properties. The crystals were grown by the hanging-drop vapour-diffusion method and belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 83.2, b = 164.9, c = 92.0 Å, β = 116.0°. Diffraction data were collected to 2.5 Å resolution. According to a Matthews coefficient calculation, there are four monomers in the asymmetric unit with a V M of 3.0 Å 3 Da −1 and a solvent content of 59.3%. The initial structure of BHRI has been determined by the molecular-replacement method

Memory T-cell immune response in healthy young adults vaccinated with live attenuated influenza A (H5N2) vaccine.

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Chirkova, T V; Naykhin, A N; Petukhova, G D; Korenkov, D A; Donina, S A; Mironov, A N; Rudenko, L G

2011-10-01

Cellular immune responses of both CD4 and CD8 memory/effector T cells were evaluated in healthy young adults who received two doses of live attenuated influenza A (H5N2) vaccine. The vaccine was developed by reassortment of nonpathogenic avian A/Duck/Potsdam/1402-6/68 (H5N2) and cold-adapted A/Leningrad/134/17/57 (H2N2) viruses. T-cell responses were measured by standard methods of intracellular cytokine staining of gamma interferon (IFN-γ)-producing cells and a novel T-cell recognition of antigen-presenting cells by protein capture (TRAP) assay based on the trogocytosis phenomenon, namely, plasma membrane exchange between interacting immune cells. TRAP enables the detection of activated trogocytosis-positive T cells after virus stimulation. We showed that two doses of live attenuated influenza A (H5N2) vaccine promoted both CD4 and CD8 T-memory-cell responses in peripheral blood of healthy young subjects in the clinical study. Significant differences in geometric mean titers (GMTs) of influenza A (H5N2)-specific IFN-γ(+) cells were observed at day 42 following the second vaccination, while peak levels of trogocytosis(+) T cells were detected earlier, on the 21st day after the second vaccination. The inverse correlation of baseline levels compared to postvaccine fold changes in GMTs of influenza-specific CD4 and CD8 T cells demonstrated that baseline levels of these specific cells could be considered a predictive factor of vaccine immunogenicity.

An Adjuvanted Herpes Simplex Virus 2 Subunit Vaccine Elicits a T Cell Response in Mice and Is an Effective Therapeutic Vaccine in Guinea Pigs

Science.gov (United States)

Skoberne, Mojca; Cardin, Rhonda; Lee, Alexander; Kazimirova, Ana; Zielinski, Veronica; Garvie, Danielle; Lundberg, Amy; Larson, Shane; Bravo, Fernando J.; Bernstein, David I.; Flechtner, Jessica B.

2013-01-01

Immunotherapeutic herpes simplex virus 2 (HSV-2) vaccine efficacy depends upon the promotion of antigen-specific immune responses that inhibit reactivation or reactivated virus, thus controlling both recurrent lesions and viral shedding. In the present study, a candidate subunit vaccine, GEN-003/MM-2, was evaluated for its ability to induce a broad-spectrum immune response in mice and therapeutic efficacy in HSV-2-infected guinea pigs. GEN-003 is comprised of HSV-2 glycoprotein D2 (gD2ΔTMR340-363) and a truncated form of infected cell polypeptide 4 (ICP4383-766), formulated with Matrix M-2 (MM-2) adjuvant (GEN-003/MM-2). In addition to eliciting humoral immune responses, CD4+ and CD8+ T cells characterized by the secretion of multiple cytokines and cytolytic antigen-specific T cell responses that were able to be recalled at least 44 days after the last immunization were induced in immunized mice. Furthermore, vaccination with either GEN-003 or GEN-003/MM-2 led to significant reductions in both the prevalence and severity of lesions in HSV-2-infected guinea pigs compared to those of phosphate-buffered saline (PBS) control-vaccinated animals. While vaccination with MM-2 adjuvant alone decreased recurrent disease symptoms compared to the PBS control group, the difference was not statistically significant. Importantly, the frequency of recurrent viral shedding was considerably reduced in GEN-003/MM-2-vaccinated animals but not in GEN-003- or MM-2-vaccinated animals. These findings suggest a possible role for immunotherapeutic GEN-003/MM-2 vaccination as a viable alternative to chronic antiviral drugs in the treatment and control of genital herpes disease. PMID:23365421

A recombinant modified vaccinia ankara vaccine encoding Epstein-Barr Virus (EBV) target antigens: a phase I trial in UK patients with EBV-positive cancer.

Science.gov (United States)

Taylor, Graham S; Jia, Hui; Harrington, Kevin; Lee, Lip Wai; Turner, James; Ladell, Kristin; Price, David A; Tanday, Manjit; Matthews, Jen; Roberts, Claudia; Edwards, Ceri; McGuigan, Lesley; Hartley, Andrew; Wilson, Steve; Hui, Edwin P; Chan, Anthony T C; Rickinson, Alan B; Steven, Neil M

2014-10-01

Epstein-Barr virus (EBV) is associated with several cancers in which the tumor cells express EBV antigens EBNA1 and LMP2. A therapeutic vaccine comprising a recombinant vaccinia virus, MVA-EL, was designed to boost immunity to these tumor antigens. A phase I trial was conducted to demonstrate the safety and immunogenicity of MVA-EL across a range of doses. Sixteen patients in the United Kingdom (UK) with EBV-positive nasopharyngeal carcinoma (NPC) received three intradermal vaccinations of MVA-EL at 3-weekly intervals at dose levels between 5 × 10(7) and 5 × 10(8) plaque-forming units (pfu). Blood samples were taken at screening, after each vaccine cycle, and during the post-vaccination period. T-cell responses were measured using IFNγ ELISpot assays with overlapping EBNA1/LMP2 peptide mixes or HLA-matched epitope peptides. Polychromatic flow cytometry was used to characterize functionally responsive T-cell populations. Vaccination was generally well tolerated. Immunity increased after vaccination to at least one antigen in 8 of 14 patients (7/14, EBNA1; 6/14, LMP2), including recognition of epitopes that vary between EBV strains associated with different ethnic groups. Immunophenotypic analysis revealed that vaccination induced differentiation and functional diversification of responsive T-cell populations specific for EBNA1 and LMP2 within the CD4 and CD8 compartments, respectively. MVA-EL is safe and immunogenic across diverse ethnicities and thus suitable for use in trials against different EBV-positive cancers globally as well as in South-East Asia where NPC is most common. The highest dose (5 × 10(8) pfu) is recommended for investigation in current phase IB and II trials. ©2014 American Association for Cancer Research.

Fractional Dosing of Yellow Fever Vaccine to Extend Supply: A Modeling Study

Science.gov (United States)

Peak, Corey M.; Leung, Gabriel M.

2016-01-01

Background The ongoing yellow fever (YF) epidemic in Angola strains the global vaccine supply, prompting WHO to adopt dose sparing for its vaccination campaign in Kinshasa in July–August 2016. Although a 5-fold fractional-dose vaccine is similar to standard-dose vaccine in safety and immunogenicity, efficacy is untested. There is an urgent need to ensure the robustness of fractional-dose vaccination by elucidating the conditions under which dose fractionation would reduce transmission. Methods We estimate the effective reproductive number for YF in Angola using disease natural history and case report data. With simple mathematical models of YF transmission, we calculate the infection attack rate (IAR, the proportion of population infected over the course of an epidemic) under varying levels of transmissibility and five-fold fractional-dose vaccine efficacy for two vaccination scenarios: (i) random vaccination in a hypothetical population that is completely susceptible; (ii) the Kinshasa vaccination campaign in July–August 2016 with different age cutoff for fractional-dose vaccines. Findings We estimate the effective reproductive number early in the Angola outbreak was between 5·2 and 7·1. If vaccine action is all-or-nothing (i.e. a proportion VE of vaccinees receives complete and the remainder receive no protection), n-fold fractionation can dramatically reduce IAR as long as efficacy VE exceeds 1/n. This benefit threshold becomes more stringent if vaccine action is leaky (i.e. the susceptibility of each vaccinee is reduced by a factor that is equal to the vaccine efficacy VE). The age cutoff for fractional-dose vaccines chosen by the WHO for the Kinshasa vaccination campaign (namely, 2 years) provides the largest reduction in IAR if the efficacy of five-fold fractional-dose vaccines exceeds 20%. Interpretation Dose fractionation is a very effective strategy for reducing infection attack rate that would be robust with a large margin for error in case

Reactogenicity and immunogenicity of inactivated poliovirus vaccine produced from Sabin strains: a phase I Trial in healthy adults in Cuba.

Science.gov (United States)

Resik, Sonia; Tejeda, Alina; Fonseca, Magilé; Alemañi, Nilda; Diaz, Manuel; Martinez, Yenisleidys; Garcia, Gloria; Okayasu, Hiromasa; Burton, Anthony; Bakker, Wilfried A M; Verdijk, Pauline; Sutter, Roland W

2014-09-22

To ensure that developing countries have the option to produce inactivated poliovirus vaccine (IPV), the Global Polio Eradication Initiative has promoted the development of an IPV using Sabin poliovirus strains (Sabin IPV). This trial assessed the reactogenicity and immunogenicity of Sabin IPV and adjuvanted Sabin IPV in healthy adults in Cuba. This is a randomized, controlled phase I trial, enrolling 60 healthy (previously vaccinated) male human volunteers, aged 19-23 years to receive one dose of either Sabin IPV (20:32:64 DU/dose), adjuvanted Sabin IPV (10:16:32 DU/dose), or conventional Salk IPV (40:8:32 DU/dose). The primary endpoint for reactogenicity relied on monitoring of adverse events. The secondary endpoint measured boosting immune responses (i.e. seroconversion or 4-fold rise) of poliovirus antibody, assessed by neutralization assays. Sixty subjects fulfilled the study requirements. No serious adverse events reported were attributed to trial interventions during the 6-month follow-up period. Twenty-eight days after vaccination, boosting immune responses against poliovirus types 1-3 were between 90% and 100% in all vaccination groups. There was a more than 6-fold increase in median antibody titers between pre- and post-vaccination titers in all vaccination groups. Both Sabin IPV and adjuvanted Sabin IPV were well tolerated and immunogenic against all poliovirus serotypes. This result suggests that the aluminum adjuvant may allow a 50% (or higher) dose reduction. Copyright © 2014. Published by Elsevier Ltd.

Understanding how different recruitment strategies impact parent engagement with an iPad-based intervention to provide personalized information about adolescent vaccines.

Science.gov (United States)

Dempsey, Amanda F; Maertens, Julie; Beaty, Brenda L; O'Leary, Sean T

2015-05-01

Inadequate provider time for addressing parents' questions and concerns about adolescent vaccines is a barrier to vaccine utilization. We sought to determine how different recruitment strategies impact the degree of engagement with an intervention that provided this information via an iPad placed in a clinical setting. We provided to three pediatric practices in the Denver area the "Teen VaxScene" web site that generates individually customized information for parents about adolescent vaccines. Three recruitment strategies were assessed for their impact on parental use of the intervention as follows: passive recruitment using posters to advertise a "kiosk" version of the intervention; posters plus a $10 incentive for using the kiosk; and posters plus a $10 incentive plus decoupling the iPad from the kiosks to enable "roving." We assessed the engagement with the intervention at multiple levels including log in, consent, and completion of a baseline survey and viewing individually tailored web pages. Surveys were used to assess barriers to using the intervention. During the 14-month study period, 693 people had contact with the iPad, 199 consented, and 48 completed the survey to enable creation of tailored content; and 42 used the tailored site. Five times as many parents (n = 40) consented to participation during the 2 months when the intervention was "roving" than during the 10-month "passive" recruitment period. Engagement with the tailored material was low, with most users viewing only the "table of contents" pages. Utilizers and nonutilizers of the intervention had similar demographic characteristics. Enabling the iPad to "rove" in the clinic greatly increased the proportion of parents consenting to use the intervention. However, meaningful engagement with the material was low. Further research is needed to understand the most effective and time efficient ways to provide vaccine-related educational information to parents of adolescents. Copyright © 2015 Society

[Historical development of vaccines. Introduction: Hazards and rationality in the vaccinal approach].

Science.gov (United States)

Moulin, A M

1995-01-01

The aim of this paper is to introduce the one hundred years of vaccination that has passed since Louis Pasteur first coined this generic term. According to the late Jonas Salk, vaccinology is a science encompassing all aspects of vaccine from its conception in the laboratory to its production by companies and its application and distribution in the field. In this historical survey I explore how vaccination never consisted of a simple and uniform application of a rational model, but rather diverged along various pathways, several of which were discarded in retrospect as being hazardous, and I analyse the ongoing interplay between rational and inventive thinking.

Health and economic impact of HPV 16/18 vaccination and cervical cancer screening in Eastern Africa.

Science.gov (United States)

Campos, Nicole G; Kim, Jane J; Castle, Philip E; Ortendahl, Jesse D; O'Shea, Meredith; Diaz, Mireia; Goldie, Sue J

2012-06-01

Eastern Africa has the world's highest cervical cancer incidence and mortality rates. We used epidemiologic data from Kenya, Mozambique, Tanzania, Uganda, and Zimbabwe to develop models of HPV-related infection and disease. For each country, we assessed HPV vaccination of girls before age 12 followed by screening with HPV DNA testing once, twice, or three times per lifetime (at ages 35, 40, 45). For women over age 30, we assessed only screening (with HPV DNA testing up to three times per lifetime or VIA at age 35). Assuming no waning immunity, mean reduction in lifetime cancer risk associated with vaccination ranged from 36 to 45%, and vaccination followed by screening once per lifetime at age 35 with HPV DNA testing ranged from 43 to 51%. For both younger and older women, the most effective screening strategy was HPV DNA testing three times per lifetime. Provided the cost per vaccinated girl was less than I$10 (I$2 per dose), vaccination had an incremental cost-effectiveness ratio [I$ (international dollars)/year of life saved (YLS)] less than the country-specific per capita GDP, a commonly cited heuristic for "very cost-effective" interventions. If the cost per vaccinated girl was between I$10 (I$2 per dose) and I$25 (I$5 per dose), vaccination followed by HPV DNA testing would save the most lives and would be considered good value for public health dollars. These results should be used to catalyze design and evaluation of HPV vaccine delivery and screening programs, and contribute to a dialogue on financing HPV vaccination in poor countries. Copyright © 2011 UICC.

CHARACTERIZATION OF A NEW BACILLUS-STEAROTHERMOPHILUS ISOLATE - A HIGHLY THERMOSTABLE ALPHA-AMYLASE-PRODUCING STRAIN

NARCIS (Netherlands)

WIND, RD; BUITELAAR, RM; EGGINK, G; HUIZING, HJ; DIJKHUIZEN, L

A novel strain of Bacillus stearothermophilus was isolated from samples of a potato-processing industry. Compared to known alpha-amylases from other B. stearothermophilus strains, the isolate was found to produce a highly thermostable alpha-amylase. The half-time of inactivation of this

Thermostable Cellulases: Why & How?

Energy Technology Data Exchange (ETDEWEB)

Kumar, Manoj [Royal DSM, San Francisco, CA (United States)

2010-03-24

These are a set of slides from the conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

Thermostable Cellulases: Why & How?

Energy Technology Data Exchange (ETDEWEB)

Kumar, Manoj [DSM Innovation, Incorporated, San Francisco, CA (United States)

2010-04-19

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

What Vaccinations Does My Child Need?

Science.gov (United States)

... needed before your child can enter school or childcare. How do vaccines work? Vaccines help your child’s ... routine vaccines is covered by your health insurance company. How do I know when my child should ...

Field Efficacy of an Attenuated Infectious Bronchitis Variant 2 Virus Vaccine in Commercial Broiler Chickens

Directory of Open Access Journals (Sweden)

Mohamed A. Elhady

2018-05-01

Full Text Available Egyptian poultry suffer from frequent respiratory disease outbreaks associated with Infectious Bronchitis Virus (IBV variant 2 strains (Egy/VarII. Different vaccination programs using imported vaccines have failed to protect the flocks from field challenge. Recent studies confirmed a successful protection using homologous strains as live attenuated vaccines. In this study, a newly developed live attenuated IB-VAR2 vaccine representing the GI-23 Middle East IBV lineage was evaluated in day-old commercial broilers in an IBV-endemic area. A commercial broiler flock was vaccinated with the IB-VAR2 vaccine at day-old age followed by IB-H120 at day 16. The vaccinated flock was monitored on a weekly basis till the slaughter age. The health status and growth performance were monitored, and selected viral pathogen real-time RT-PCR (rRT-PCR detection was conducted on a weekly basis. Finally, the flock was compared to a nearby farm with only the classical IB-H120 vaccination program. Results showed that the IB-VAR2 vaccine was tolerable in day-old broiler chicks. The IBV virus rRT-PCR detection was limited to the trachea as compared to its nephropathogenic parent virus. Respiratory disease problems and high mortalities were reported in the IB-H120-only vaccinated flock. An exposure to a wild-type Egy/VarII strain was confirmed in both flocks as indicated by partial IBV S1 gene sequence. Even though the IB-VAR2-vaccinated flock performance was better than the flock that received only IB-H120, the IBV ELISA (enzyme-linked immunosorbent assay and log2 Haemagglutination inhibition (HI antibody mean titers remained high (3128 ± 2713 and ≥9 log2, respectively until the 28th day of age. The current study demonstrates the safety and effectiveness of IB-VAR2 as a live attenuated vaccine in day-old commercial broilers. Also, the combination of IB-VAR2 and classical IBV vaccines confers a broader protective immune response against IBV in endemic areas.

Biologic interactions between HSV-2 and HIV-1 and possible implications for HSV vaccine development.

Science.gov (United States)

Schiffer, Joshua T; Gottlieb, Sami L

2017-09-25

Development of a safe and effective vaccine against herpes simplex virus type 2 (HSV-2) has the potential to limit the global burden of HSV-2 infection and disease, including genital ulcer disease and neonatal herpes, and is a global sexual and reproductive health priority. Another important potential benefit of an HSV-2 vaccine would be to decrease HIV infections, as HSV-2 increases the risk of HIV-1 acquisition several-fold. Acute and chronic HSV-2 infection creates ulcerations and draws dendritic cells and activated CD4+ T cells into genital mucosa. These cells are targets for HIV entry and replication. Prophylactic HSV-2 vaccines (to prevent infection) and therapeutic vaccines (to modify or treat existing infections) are currently under development. By preventing or modifying infection, an effective HSV-2 vaccine could limit HSV-associated genital mucosal inflammation and thus HIV risk. However, a vaccine might have competing effects on HIV risk depending on its mechanism of action and cell populations generated in the genital mucosa. In this article, we review biologic interactions between HSV-2 and HIV-1, consider HSV-2 vaccine development in the context of HIV risk, and discuss implications and research needs for future HSV vaccine development. Copyright © 2017 Elsevier Ltd. All rights reserved.

Nasal delivery of an adenovirus-based vaccine bypasses pre-existing immunity to the vaccine carrier and improves the immune response in mice.

Directory of Open Access Journals (Sweden)

Maria A Croyle

Full Text Available Pre-existing immunity to human adenovirus serotype 5 (Ad5 is common in the general population. Bypassing pre-existing immunity could maximize Ad5 vaccine efficacy. Vaccination by the intramuscular (I.M., nasal (I.N. or oral (P.O. route with Ad5 expressing Ebola Zaire glycoprotein (Ad5-ZGP fully protected naïve mice against lethal challenge with Ebola. In the presence of pre-existing immunity, only mice vaccinated I.N. survived. The frequency of IFN-gamma+ CD8+ T cells was reduced by 80% and by 15% in animals vaccinated by the I.M. and P.O. routes respectively. Neutralizing antibodies could not be detected in serum from either treatment group. Pre-existing immunity did not compromise the frequency of IFN-gamma+ CD8+ T cells (3.9+/-1% naïve vs. 3.6+/-1% pre-existing immunity, PEI nor anti-Ebola neutralizing antibody (NAB, 40+/-10 reciprocal dilution, both groups. The number of INF-gamma+ CD8+ cells detected in bronchioalveolar lavage fluid (BAL after I.N. immunization was not compromised by pre-existing immunity to Ad5 (146+/-14, naïve vs. 120+/-16 SFC/million MNCs, PEI. However, pre-existing immunity reduced NAB levels in BAL by approximately 25% in this group. To improve the immune response after oral vaccination, the Ad5-based vaccine was PEGylated. Mice given the modified vaccine did not survive challenge and had reduced levels of IFN-gamma+ CD8+ T cells 10 days after administration (0.3+/-0.3% PEG vs. 1.7+/-0.5% unmodified. PEGylation did increase NAB levels 2-fold. These results provide some insight about the degree of T and B cell mediated immunity necessary for protection against Ebola virus and suggest that modification of the virus capsid can influence the type of immune response elicited by an Ad5-based vaccine.

Evaluation of two vaccine education interventions to improve pertussis vaccination among pregnant African American women: A randomized controlled trial.

Science.gov (United States)

Kriss, Jennifer L; Frew, Paula M; Cortes, Marielysse; Malik, Fauzia A; Chamberlain, Allison T; Seib, Katherine; Flowers, Lisa; Ault, Kevin A; Howards, Penelope P; Orenstein, Walter A; Omer, Saad B

2017-03-13

Vaccination coverage with tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis (Tdap) vaccine in pregnancy or immediately postpartum has been low. Limited data exist on rigorously evaluated interventions to increase maternal vaccination, including Tdap. Tailored messaging based on the Elaboration Likelihood Model (ELM) framework has been successful in improving uptake of some public health interventions. We evaluated the effect of two ELM-based vaccine educational interventions on Tdap vaccination among pregnant African American women, a group of women who tend to have lower vaccine uptake compared with other groups. We conducted a prospective randomized controlled trial to pilot test two interventions - an affective messaging video and a cognitive messaging iBook - among pregnant African American women recruited during routine prenatal care visits. We measured Tdap vaccination during the perinatal period (during pregnancy and immediately postpartum), reasons for non-vaccination, and intention to receive Tdap in the next pregnancy. Among the enrolled women (n=106), 90% completed follow-up. Tdap vaccination in the perinatal period was 18% in the control group; 50% in the iBook group (Risk Ratio [vs. control group]: 2.83; 95% CI, 1.26-6.37), and 29% in the video group (RR: 1.65; 95% CI, 0.66-4.09). From baseline to follow-up, women's reported intention to receive Tdap during the next pregnancy improved in all three groups. Among unvaccinated women, the most common reason reported for non-vaccination was lack of a recommendation for Tdap by the woman's physician. Education interventions that provide targeted information for pregnant women in an interactive manner may be useful to improve Tdap vaccination during the perinatal period. However, larger studies including multiple racial and ethnic groups are needed to evaluate robustness of our findings. clinicaltrials.gov Identifier: NCT01740310. Copyright © 2017 Elsevier Ltd. All rights reserved.

Asthmatic Children And Immunological Effects Of BCG Vaccine Key words: Asthmatic children, BCG vaccine

International Nuclear Information System (INIS)

Saaed, A.I.

2011-01-01

A TH2 screwed immune response is known to play a crucial role in the pathogenesis of allergy, so, preventing the differentiation of TH cells. The TH2 cells are appeared as a logical therapeutic approach to atopic asthma. The purpose of TH1 study was to determine the possible role of BCG vaccine on asthma and whether a TH1 type immune response elicited by BCG immunization could suppress the allergic sensitization in childhood asthma. Seventy asthmatic patients (50 atopic and 20 non-atopic) and fifty healthy individuals were subjected to TH1 study. Tuberculin test was performed for all groups then subjects with positive tuberculin test were excluded. The BCG vaccine was given for all groups with assessment of TH1 and TH2 cytokine response by measuring total IgE, IL-4 (for TH2 response) and INF-γ (for TH1 response). Significant reduction in IgE and IL-4, and elevation in INF-γ were determined in group I (atopic asthma) following BCG vaccination. There was non-significant change observed in IgE and IL-4 levels of group II while significant reduction in IL-4 and significant increase in INF-γ was observed after BCG vaccine

Doubling Power Output of Starch Biobattery Treated by the Most Thermostable Isoamylase from an Archaeon Sulfolobus tokodaii.

Science.gov (United States)

Cheng, Kun; Zhang, Fei; Sun, Fangfang; Chen, Hongge; Percival Zhang, Y-H

2015-08-20

Biobattery, a kind of enzymatic fuel cells, can convert organic compounds (e.g., glucose, starch) to electricity in a closed system without moving parts. Inspired by natural starch metabolism catalyzed by starch phosphorylase, isoamylase is essential to debranch alpha-1,6-glycosidic bonds of starch, yielding linear amylodextrin - the best fuel for sugar-powered biobattery. However, there is no thermostable isoamylase stable enough for simultaneous starch gelatinization and enzymatic hydrolysis, different from the case of thermostable alpha-amylase. A putative isoamylase gene was mined from megagenomic database. The open reading frame ST0928 from a hyperthermophilic archaeron Sulfolobus tokodaii was cloned and expressed in E. coli. The recombinant protein was easily purified by heat precipitation at 80 (o)C for 30 min. This enzyme was characterized and required Mg(2+) as an activator. This enzyme was the most stable isoamylase reported with a half lifetime of 200 min at 90 (o)C in the presence of 0.5 mM MgCl2, suitable for simultaneous starch gelatinization and isoamylase hydrolysis. The cuvett-based air-breathing biobattery powered by isoamylase-treated starch exhibited nearly doubled power outputs than that powered by the same concentration starch solution, suggesting more glucose 1-phosphate generated.

Continued Follow-Up of Phambili Phase 2b Randomized HIV-1 Vaccine Trial Participants Supports Increased HIV-1 Acquisition among Vaccinated Men.

Directory of Open Access Journals (Sweden)

Zoe Moodie

Full Text Available The Phase 2b double-blinded, randomized Phambili/HVTN 503 trial evaluated safety and efficacy of the MRK Ad5 gag/pol/nef subtype B HIV-1 preventive vaccine vs placebo in sexually active HIV-1 seronegative participants in South Africa. Enrollment and vaccinations stopped and participants were unblinded but continued follow-up when the Step study evaluating the same vaccine in the Americas, Caribbean, and Australia was unblinded for non-efficacy. Final Phambili analyses found more HIV-1 infections amongst vaccine than placebo recipients, impelling the HVTN 503-S recall study.HVTN 503-S sought to enroll all 695 HIV-1 uninfected Phambili participants, provide HIV testing, risk reduction counseling, physical examination, risk behavior assessment and treatment assignment recall. After adding HVTN 503-S data, HIV-1 infection hazard ratios (HR vaccine vs. placebo were estimated by Cox models.Of the 695 eligible, 465 (67% enrolled with 230 from the vaccine group and 235 from the placebo group. 38% of the 184 Phambili dropouts were enrolled. Enrollment did not differ by treatment group, gender, or baseline HSV-2. With the additional 1286 person years of 503-S follow-up, the estimated HR over Phambili and HVTN 503-S follow-up was 1.52 (95% CI 1.08-2.15, p = 0.02, 82 vaccine/54 placebo infections. The HR was significant for men (HR = 2.75, 95% CI 1.49, 5.06, p = 0.001 but not for women (HR = 1.12, 95% CI 0.73, 1.72, p = 0.62.The additional follow-up from HVTN 503-S supported the Phambili finding of increased HIV-1 acquisition among vaccinated men and strengthened the evidence of lack of vaccine effect among women.clinicaltrials.gov NCT00413725 SA National Health Research Database DOH-27-0207-1539.

Yellow Fever Vaccine: What You Need to Know

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... How can I prevent yellow fever? Yellow fever vaccine Yellow fever vaccine can prevent yellow fever. Yellow fever vaccine ... such as those containing DEET. 3 Yellow fever vaccine Yellow fever vaccine is a live, weakened virus. It is ...

Avian metapneumovirus M2:2 protein inhibits replication in Vero cells: modification facilitates live vaccine development.

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Clubbe, Jayne; Naylor, Clive J

2011-11-28

Throughout the world, avian metapneumovirus (AMPV) infection of subtype A is principally controlled by two live vaccines both derived from UK field strain #8544. Improvements of those vaccines by use of reverse genetics technology was found to be hampered by the inability of #8544 to replicate in the commonly exploited Vero cell based reverse genetics system. A systematic reverse genetics based genome modification of a DNA copy of #8544, employing sequence data from a Vero grown, #8544 derived, live vaccine; was used to determine mutations required to facilitate virus recovery and replication in Vero cells. This identified a single coding substitution in the M2:2 reading frame as responsible. Furthermore, ablation of M2:2 was found to elicit the same outcome. M2:2 sequence analysis of seven AMPVs found Vero cell adaption to be associated with non similar amino acid changes in M2:2. The study shows that M2:2 modification of field virus #8544 will enable research leading to improved vaccines. This may have more general application to other AMPV field strains. Copyright © 2011 Elsevier Ltd. All rights reserved.

Vaccine profile of herpes zoster (HZ/su) subunit vaccine.

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Cunningham, Anthony L; Heineman, Thomas

2017-07-01

Herpes zoster (HZ) causes an often severe and painful rash in older people and may be complicated by prolonged pain (postherpetic neuralgia; PHN) and by dissemination in immune-compromised patients. HZ results from reactivation of latent varicella-zoster virus (VZV) infection, often associated with age-related or other causes of decreased T cell immunity. A live attenuated vaccine boosts this immunity and provides partial protection against HZ, but this decreases with age and declines over 8 years. Areas covered: A new HZ subunit (HZ/su) vaccine combines a key surface VZV glycoprotein (E) with a T cell-boosting adjuvant system (AS01 B ) and is administered by two intramuscular injections two months apart. Expert commentary: HZ/su showed excellent efficacy of ~90% in immunocompetent adults ≥50 and ≥70 years of age, respectively, in the ZOE-50 and ZOE-70 phase III controlled trials. Efficacy was unaffected by advancing age and persisted for >3 years. Approximately 9.5% of subjects had severe, but transient (1-2 days) injection site pain, swelling or redness. Compliance with both vaccine doses was high (95%). The vaccine will have a major impact on HZ management. Phase I-II trials showed safety and immunogenicity in severely immunocompromised patients. Phase III trial results are expected soon.

The response of mute swans (Cygnus olor, Gm. 1789) to vaccination against avian influenza with an inactivated H5N2 vaccine.

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Dolka, Beata; Żbikowski, Artur; Dolka, Izabella; Szeleszczuk, Piotr

2016-10-22

Recent epidemics of highly pathogenic avian influenza (HPAI) produced an unprecedented number of cases in mute swans (Cygnus olor) in European countries, which indicates that these birds are very sensitive to the H5N1 virus. The HPAI outbreaks stirred a debate on the controversial stamping-out policy in populations of protected bird species. After preventive vaccination had been approved in the European Union, several countries have introduced vaccination schemes to protect poultry, captive wild birds or exotic birds in zoos against HPAI. The aim of this study was to investigate the immune response of wild mute swans to immunization with an inactivated AI H5N2 vaccine approved for use in poultry. The serological responses of mute swans were assessed by comparison with racing pigeons (Columba livia), a species which is characterized by different susceptibility to infection with the H5N1 HPAI virus and plays a questionable role in the ecology of influenza (H5N1) viruses. Swans were vaccinated once or twice at an interval of 4 weeks. The humoral immune response was evaluated by hemagglutination inhibition (HI) and NP-ELISA. The lymphocyte blast transformation test was used to determine the cell-mediated immune response. Higher values of the geometric mean titer (GMT) and 100 % seroconversion (HI ≥32) were noted in double vaccinated swans (1448.2) than in single vaccinated swans (128.0) or in double vaccinated pigeons (215.3). Significant differences in HI titers were observed between swans and pigeons, but no variations in ELISA scores were noted after the booster dose. Immunization of swans had no effect on the proliferative activity of lymphocytes. The inactivated H5N2 vaccine was safe and immunogenic for mute swans and pigeons. Vaccination may have practical implications for swans kept in zoos, wildlife parks or rehabilitation centers. However, challenge studies are needed to prove the efficacy of the H5N2 AI vaccine.

Characterization of an extremely salt-tolerant and thermostable phytase from Bacillus amyloliquefaciens US573.

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Boukhris, Ines; Farhat-Khemakhem, Ameny; Blibech, Monia; Bouchaala, Kameleddine; Chouayekh, Hichem

2015-09-01

The extracellular phytase produced by the Bacillus amyloliquefaciens US573 strain, isolated from geothermal soil located in Southern Tunisia was purified and characterized. This calcium-dependent and bile-stable enzyme (PHY US573) was optimally active at pH 7.5 and 70 °C. It showed a good stability at pH ranging from 4 to 10, and especially, an exceptional thermostability as it recovered 50 and 62% of activity after heating for 10 min at 100 and 90 °C, respectively. In addition, PHY US573 was found to be extremely salt-tolerant since it preserved 80 and 95% of activity in the presence of 20 g/l of NaCl and LiCl, respectively. The gene corresponding to PHY US573 was cloned. It encodes a 383 amino acids polypeptide exhibiting 99% identity with the highly thermostable phytases from Bacillus sp. MD2 and B. amyloliquefaciens DS11 (3 and 5 residues difference, respectively), suggesting the existence of common molecular determinants responsible for their remarkable heat stability. Overall, our findings illustrated that in addition to its high potential for application in feed industry, the salt tolerance of the PHY US573 phytase, may represent an exciting new avenue for improvement of phosphorus-use efficiency of salt-tolerant plants in soils with high salt and phytate content. Copyright © 2015 Elsevier B.V. All rights reserved.

Protective efficacy of an inactivated vaccine against H9N2 avian influenza virus in ducks.

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Teng, Qiaoyang; Shen, Weixia; Liu, Qinfang; Rong, Guangyu; Chen, Lin; Li, Xuesong; Chen, Hongjun; Yang, Jianmei; Li, Zejun

2015-09-17

Wild ducks play an important role in the evolution of avian influenza viruses (AIVs). Domestic ducks in China are known to carry and spread H9N2 AIVs that are thought to have contributed internal genes for the recent outbreak of zoonotic H7N9 virus. In order to protect animal and public health, an effective vaccine is urgently needed to block and prevent the spread of H9N2 virus in ducks. We developed an inactivated H9N2 vaccine (with adjuvant Montanide ISA 70VG) based on an endemic H9N2 AIV and evaluated this vaccine in ducks. The results showed that the inactivated H9N2 vaccine was able to induce a strong and fast humoral immune response in vaccinated ducks. The hemagglutination inhibition titer in the sera increased fast, and reached its peak of 12.3 log2 at 5 weeks post-vaccination in immunized birds and remained at a high level for at least 37 weeks post-vaccination. Moreover, viral shedding was completely blocked in vaccinated ducks after challenge with a homologous H9N2 AIV at both 3 and 37 weeks post-vaccination. The results of this study indicate that the inactivated H9N2 vaccine induces high and prolonged immune response in vaccinated ducks and are efficacious in protecting ducks from H9N2 infection.

Vaccine Adjuvants in Fish Vaccines Make a Difference: Comparing Three Adjuvants (Montanide ISA763A Oil, CpG/Poly I:C Combo and VHSV Glycoprotein Alone or in Combination Formulated with an Inactivated Whole Salmonid Alphavirus Antigen

Directory of Open Access Journals (Sweden)

Hanna L. Thim

2014-03-01

Full Text Available Most commercial vaccines offered to the aquaculture industry include inactivated antigens (Ag formulated in oil adjuvants. Safety concerns are related to the use of oil adjuvants in multivalent vaccines for fish, since adverse side effects (e.g., adhesions can appear. Therefore, there is a request for vaccine formulations for which protection will be maintained or improved, while the risk of side effects is reduced. Here, by using an inactivated salmonid alphavirus (SAV as the test Ag, the combined use of two Toll-like receptor (TLR ligand adjuvants, CpG oligonucleotides (ODNs and poly I:C, as well as a genetic adjuvant consisting of a DNA plasmid vector expressing the viral haemorrhagic septicaemia virus (VHSV glycoprotein (G was explored. VHSV-G DNA vaccine was intramuscularly injected in combination with intraperitoneal injection of either SAV Ag alone or combined with the oil adjuvant, Montanide ISA763, or the CpG/polyI:C combo. Adjuvant formulations were evaluated for their ability to boost immune responses and induce protection against SAV in Atlantic salmon, following cohabitation challenge. It was observed that CpG/polyI:C-based formulations generated the highest neutralizing antibody titres (nAbs before challenge, which endured post challenge. nAb responses for VHSV G-DNA- and oil-adjuvanted formulations were marginal compared to the CpG/poly I:C treatment. Interestingly, heat-inactivated sera showed reduced nAb titres compared to their non-heated counterparts, which suggests a role of complement-mediated neutralization against SAV. Consistently elevated levels of innate antiviral immune genes in the CpG/polyI:C injected groups suggested a role of IFN-mediated responses. Co-delivery of the VHSV-G DNA construct with either CpG/polyI:C or oil-adjuvanted SAV vaccine generated higher CD4 responses in head kidney at 48 h compared to injection of this vector or SAV Ag alone. The results demonstrate that a combination of pattern recognizing

Post-marketing safety surveillance for inactivated and live-attenuated Japanese encephalitis vaccines in China, 2008-2013.

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Wu, Wendi; Liu, Dawei; Li, Keli; Nuorti, J Pekka; Nohynek, Hanna M; Xu, Disha; Ye, Jiakai; Zheng, Jingshan; Wang, Huaqing

2017-06-22

Two types of Japanese encephalitis (JE) vaccines, inactivated JE vaccine (JE-I) and live-attenuated JE vaccine (JE-L), are available and used in China. In particular, one JE-L, produced by a domestic manufacturer in China, was prequalified by WHO in 2013. We assessed the safety of JE vaccines in China during 2008-2013 using the Chinese National Adverse Events Following Immunization Information System (CNAEFIS) data. We retrieved AEFI reporting data about JE vaccines from CNAEFIS, 2008-2013, examined demographic characteristics of AEFI cases, and used administrative data on vaccine doses as denominator to calculate and compare crude reporting rates. We also used disproportionality reporting analysis between JE-I and JE-L to assess potential safety signals. A total of 34,879 AEFIs related with JE-I and JE-L were reported, with a ratio of male to female as 1.3:1; 361 (1.0%) cases were classified as serious. JE vaccines were administered concurrently with one or more other vaccines in 13,592 (39.0%) of cases. The overall AEFI reporting rates were 214.4 per million vaccination doses for JE-L and 176.9 for JE-I (rate ratio [RR]: 1.2, 95% confidence interval [CI]: 1.1-1.3) in 2010-2013. Febrile convulsions (FC) following JE-I was found as a signal of disproportionate reporting (SDR). However, there was no significant difference between the reporting rates of FC of JE-I and JE-L (0.3 per million vaccination doses for JE-L, 0.4 for JE-I, p=0.05). While our analysis did not find apparent safety concern of JE vaccines in China, further study should consider JE-I vaccines and febrile convulsion, and taking more sensitive methods to detect signals. Copyright © 2017. Published by Elsevier Ltd.

A phase I double blind, placebo-controlled, randomized study of a multigenic HIV-1 adenovirus subtype 35 vector vaccine in healthy uninfected adults.

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Michael C Keefer

Full Text Available We conducted a phase I, randomized, double-blind, placebo-controlled trial to assess the safety and immunogenicity of escalating doses of two recombinant replication defective adenovirus serotype 35 (Ad35 vectors containing gag, reverse transcriptase, integrase and nef (Ad35-GRIN and env (Ad35-ENV, both derived from HIV-1 subtype A isolates. The trial enrolled 56 healthy HIV-uninfected adults.Ad35-GRIN/ENV (Ad35-GRIN and Ad35-ENV mixed in the same vial in equal proportions or Ad35-GRIN was administered intramuscularly at 0 and 6 months. Participants were randomized to receive either vaccine or placebo (10/4 per group, respectively within one of four dosage groups: Ad35-GRIN/ENV 2×10(9 (A, 2×10(10 (B, 2×10(11 (C, or Ad35-GRIN 1×10(10 (D viral particles.No vaccine-related serious adverse event was reported. Reactogenicity events reported were dose-dependent, mostly mild or moderate, some severe in Group C volunteers, all transient and resolving spontaneously. IFN-γ ELISPOT responses to any vaccine antigen were detected in 50, 56, 70 and 90% after the first vaccination, and in 75, 100, 88 and 86% of Groups A-D vaccine recipients after the second vaccination, respectively. The median spot forming cells (SFC per 10(6 PBMC to any antigen was 78-139 across Groups A-C and 158-174 in Group D, after each of the vaccinations with a maximum of 2991 SFC. Four to five HIV proteins were commonly recognized across all the groups and over multiple timepoints. CD4+ and CD8+ T-cell responses were polyfunctional. Env antibodies were detected in all Group A-C vaccinees and Gag antibodies in most vaccinees after the second immunization. Ad35 neutralizing titers remained low after the second vaccination.Ad35-GRIN/ENV reactogenicity was dose-related. HIV-specific cellular and humoral responses were seen in the majority of volunteers immunized with Ad35-GRIN/ENV or Ad35-GRIN and increased after the second vaccination. T-cell responses were broad and polyfunctional

Engineering of an Extremely Thermostable Alpha/Beta Barrel Scaffold to Serve as a High Affinity Molecular Recognition Element for Use in Sensor Applications

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2015-12-23

Molecular Recognition Element For Use in Sensor Applications Report Title The overall goal of the project was to evolve a highly thermostable enzyme ( alcohol ...SECURITY CLASSIFICATION OF: The overall goal of the project was to evolve a highly thermostable enzyme ( alcohol dehydrogenase D (AdhD) from Pyrococcus...furiosus) to bind an explosive molecule, RDX. The enzyme naturally catalyzes the nicotinamide cofactor-dependent oxidation or reduction of alcohols

Evaluation of enhanced thermostability and operational stability of carbonic anhydrase from Micrococcus species.

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Bhattacharya, Abhishek; Shrivastava, Ankita; Sharma, Anjana

2013-06-01

Carbonic anhydrase (CA) was purified from Micrococcus lylae and Micrococcus luteus with 49.90 and 53.8 % yield, respectively, isolated from calcium carbonate kilns. CA from M. lylae retained 80 % stability in the pH and temperature range of 6.0-8.0 and 35-45 °C, respectively. However, CA from M. luteus was stable in the pH and temperature range of 7.5-10.0 and 35-55 °C, respectively. Cross-linked enzyme aggregates (CLEAs) raised the transition temperature of M. lylae and M. luteus CA up to 67.5 and 74.0 °C, while the operational stability (T(1/20) of CA at 55 °C was calculated to be 7.7 and 12.0 h, respectively. CA from both the strains was found to be monomeric in nature with subunit molecular weight and molecular mass of 29 kDa. Ethoxozolamide was identified as the most potent inhibitor based on both IC(50) values and inhibitory constant measurement (K(i)). The K(m) and V(max) for M. lylae CA (2.31 mM; 769.23 μmol/mg/min) and M. luteus CA (2.0 mM; 1,000 μmol/mg/min) were calculated from Lineweaver-Burk plots in terms of esterase activity. Enhanced thermostability of CLEAs alleviates its role in operational stability for application at an on-site scrubber. The characteristic profile of purified CA from Micrococcus spp. advocates its effective application in biomimetic CO(2) sequestration.

Evaluation of an experimental and commercial state-of-the-art vaccine against enteric redmouth disease (ERM) in rainbow trout by waterborne challenge with Y. ruckeri O1 biotype 2

DEFF Research Database (Denmark)

Kragelund Strøm, Helene; Otani, Maki; Villumsen, Kasper Rømer

strain obtained from an ERM disease outbreak in a Danish trout farm. This waterborne infection model gives us the opportunity to test and evaluate the effect of commercial and experimental vaccines against Y. ruckeri O1 biotype 2. An experimental vaccine containing equal amounts of Y. ruckeri O1 biotype...... has been compared to a state-of-the-art commercial ERM immersion vaccine (AquaVac® ReleraTM). Un-vaccinated and sham vaccinated rainbow trout were included as controls. Two months post vaccination the rainbow trout were challenged in duplicate with Y. ruckeri O1 biotype 2 by bath. No effect...... of the experimental immersion or bath vaccine was observed in the present study. However, full protection was achieved with i.p. injection of the experimental vaccine (pVac® ReleraTM induced a significant, partial protection relative to the control groups (p=0.002, RPS=58...

Safety and immunogenicity of inactivated poliovirus vaccine based on Sabin strains with and without aluminum hydroxide: a phase I trial in healthy adults.

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Verdijk, Pauline; Rots, Nynke Y; van Oijen, Monique G C T; Oberste, M Steven; Boog, Claire J; Okayasu, Hiromasa; Sutter, Roland W; Bakker, Wilfried A M

2013-11-12

An inactivated poliovirus vaccine (IPV) based on attenuated poliovirus strains (Sabin-1, -2 and -3) was developed for technology transfer to manufacturers in low- and middle income countries in the context of the Global Polio Eradication Initiative. Safety and immunogenicity of the Sabin-IPV was evaluated in a double-blind, randomized, controlled, phase I 'proof-of-concept' trial. Healthy male adults received a single intramuscular injection with Sabin-IPV, Sabin-IPV adjuvanted with aluminum hydroxide or conventional IPV. Virus-neutralizing titers against both Sabin and wild poliovirus strains were determined before and 28 days after vaccination. No vaccine-related serious adverse events were observed, and all local and systemic reactions were mild or moderate and transient. In all subjects, an increase in antibody titer for all types of poliovirus (both Sabin and wild strains) was observed 28 days after vaccination. Sabin-IPV and Sabin-IPV adjuvanted with aluminum hydroxide administered as a booster dose were equally immunogenic and safe as conventional IPV. EudraCTnr: 2010-024581-22, NCT01708720. Copyright © 2013 Elsevier Ltd. All rights reserved.

An Approach for a Synthetic CTL Vaccine Design against Zika Flavivirus Using Class I and Class II Epitopes Identified by Computer Modeling

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Edecio Cunha-Neto

2017-06-01

Full Text Available The threat posed by severe congenital abnormalities related to Zika virus (ZKV infection during pregnancy has turned development of a ZKV vaccine into an emergency. Recent work suggests that the cytotoxic T lymphocyte (CTL response to infection is an important defense mechanism in response to ZKV. Here, we develop the rationale and strategy for a new approach to developing cytotoxic T lymphocyte (CTL vaccines for ZKV flavivirus infection. The proposed approach is based on recent studies using a protein structure computer model for HIV epitope selection designed to select epitopes for CTL attack optimized for viruses that exhibit antigenic drift. Because naturally processed and presented human ZKV T cell epitopes have not yet been described, we identified predicted class I peptide sequences on ZKV matching previously identified DNV (Dengue class I epitopes and by using a Major Histocompatibility Complex (MHC binding prediction tool. A subset of those met the criteria for optimal CD8+ attack based on physical chemistry parameters determined by analysis of the ZKV protein structure encoded in open source Protein Data File (PDB format files. We also identified candidate ZKV epitopes predicted to bind promiscuously to multiple HLA class II molecules that could provide help to the CTL responses. This work suggests that a CTL vaccine for ZKV may be possible even if ZKV exhibits significant antigenic drift. We have previously described a microsphere-based CTL vaccine platform capable of eliciting an immune response for class I epitopes in mice and are currently working toward in vivo testing of class I and class II epitope delivery directed against ZKV epitopes using the same microsphere-based vaccine.

Ligand binding and thermostability of different allosteric states of the insulin zinc-hexamer

DEFF Research Database (Denmark)

Huus, Kasper; Havelund, Svend; Olsen, Helle B

2006-01-01

The influence of ligand binding and conformation state on the thermostability of hexameric zinc-insulin was studied by differential scanning calorimetry (DSC). The insulin hexamer exists in equilibrium between the forms T6, T3R3, and R6. Phenolic ligands induce and stabilize the T3R3- and R6-stat...

Experiments with a homologous, inactivated canine parvovirus vaccine in vaccination programmers for dogs.

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Wilson, J H; Hermann-Dekkers, W M

1982-01-01

The significance of canine parvovirus (CPV) infections as a permanent threat susceptible dogs, in particular pups, made the authors develop three liquid homologous inactivated adjuvant CPV vaccines that were compatible with existing canine vaccines and could be incorporated in current vaccination programmes. On vaccine (Kavak Parvo) contained only the CPV component, the second product (Kavak i-LP) also contained two inactivated leptospiral antigens, and the third vaccine (Kavak i-HLP) contained in addition an inactivated canine hepatitis virus. This paper reports on the studies conducted to test the safety and efficacy of the three products. They were used as such and as diluents for freeze dried vaccines containing live attenuated measles, distemper, and hepatitis viruses. The study was performed in a breeding kennel where all dogs were free from CPV antibodies and the nonvaccinated sentinels remained so for the course of the study. All vaccines proved to be safe in dogs of all ages, including pregnant bitches. The efficacy of the CPV component was studied both by monitoring antibody titres for more than a year and by challenge exposure of some dogs to virulent CPV. The results obtained from these studies prove that the CPV component used in the three vaccines can be incorporated as indicated in the recommended canine vaccination programmes. The observations that the inactivated CPV and hepatitis components do induce an active immunity in pups that are still protected by low levels of maternally derived antibodies against these viruses, make those vaccines very suitable in breeding kennels. Additional studies on a comparative basis are being continued in edemically CPV infected breeding kennels to quantify the significance of these observations in these special conditions.

Isolation and genetic characterization of a novel 2.2.1.2a H5N1 virus from a vaccinated meat-turkeys flock in Egypt.

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Salaheldin, Ahmed H; Veits, Jutta; Abd El-Hamid, Hatem S; Harder, Timm C; Devrishov, Davud; Mettenleiter, Thomas C; Hafez, Hafez M; Abdelwhab, Elsayed M

2017-03-09

Vaccination of poultry to control highly pathogenic avian influenza virus (HPAIV) H5N1 is used in several countries. HPAIV H5N1 of clade 2.2.1 which is endemic in Egypt has diversified into two genetic clades. Clade 2.2.1.1 represents antigenic drift variants in vaccinated commercial poultry while clade 2.2.1.2 variants are detected in humans and backyard poultry. Little is known about H5N1 infection in vaccinated turkeys under field conditions. Here, we describe an HPAI H5N1 outbreak in a vaccinated meat-turkey flock in Egypt. Birds were vaccinated with inactivated H5N2 and H5N1 vaccines at 8 and 34 days of age, respectively. At 72 nd day of age (38 days post last vaccination), turkeys exhibited mild respiratory signs, cyanosis of snood and severe congestion of the internal organs. Survivors had a reduction in feed consumption and body gain. A mortality of ~29% cumulated within 10 days after the onset of clinical signs. Laboratory diagnosis using RT-qPCRs revealed presence of H5N1 but was negative for H7 and H9 subtypes. A substantial antigenic drift against different serum samples from clade 2.2.1.1 and clade 2.3.4.4 was observed. Based on full genome sequence analysis the virus belonged to clade 2.2.1.2 but clustered with recent H5N1 viruses from 2015 in poultry in Israel, Gaza and Egypt in a novel subclade designated here 2.2.1.2a which is distinct from 2014/2015 2.2.1.2 viruses. These viruses possess 2.2.1.2 clade-specific genetic signatures and also mutations in the HA similar to those in clade 2.2.1.1 that enabled evasion from humoral immune response. Taken together, this manuscript describes a recent HPAI H5N1 outbreak in vaccinated meat-turkeys in Egypt after infection with a virus representing novel distinct 2.2.1.2a subclade. Infection with HPAIV H5N1 in commercial turkeys resulted in significant morbidity and mortality despite of vaccination using H5 vaccines. The isolated virus showed antigenic drift and clustered in a novel cluster designated here

Improvement of the Immunogenicity of Porcine Circovirus Type 2 DNA Vaccine by Recombinant ORF2 Gene and CpG Motifs.

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Li, Jun; Shi, Jian-Li; Wu, Xiao-Yan; Fu, Fang; Yu, Jiang; Yuan, Xiao-Yuan; Peng, Zhe; Cong, Xiao-Yan; Xu, Shao-Jian; Sun, Wen-Bo; Cheng, Kai-Hui; Du, Yi-Jun; Wu, Jia-Qiang; Wang, Jin-Bao; Huang, Bao-Hua

2015-06-01

Nowadays, adjuvant is still important for boosting immunity and improving resistance in animals. In order to boost the immunity of porcine circovirus type 2 (PCV2) DNA vaccine, CpG motifs were inserted. In this study, the dose-effect was studied, and the immunity of PCV2 DNA vaccines by recombinant open reading frame 2 (ORF2) gene and CpG motifs was evaluated. Three-week-old Changbai piglets were inoculated intramuscularly with 200 μg, 400 μg, and 800 μg DNA vaccines containing 14 and 18 CpG motifs, respectively. Average gain and rectum temperature were recorded everyday during the experiments. Blood was collected from the piglets after vaccination to detect the changes of specific antibodies, interleukin-2, and immune cells every week. Tissues were collected for histopathology and polymerase chain reaction. The results indicated that compared to those of the control piglets, all concentrations of two DNA vaccines could induce PCV2-specific antibodies. A cellular immunity test showed that PCV2-specific lymphocytes proliferated the number of TH, TC, and CD3+ positive T-cells raised in the blood of DNA vaccine immune groups. There was no distinct pathological damage and viremia occurring in pigs that were inoculated with DNA vaccines, but there was some minor pathological damage in the control group. The results demonstrated that CpG motifs as an adjuvant could boost the humoral and cellular immunity of pigs to PCV2, especially in terms of cellular immunity. Comparing two DNA vaccines that were constructed, the one containing 18 CpG motifs was more effective. This is the first report that CpG motifs as an adjuvant insert to the PCV2 DNA vaccine could boost immunity.

Vaccine protection of chickens against antigenically diverse H5 highly pathogenic avian influenza isolates with a live HVT vector vaccine expressing the influenza hemagglutinin gene derived from a clade 2.2 avian influenza virus.

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Kapczynski, Darrell R; Esaki, Motoyuki; Dorsey, Kristi M; Jiang, Haijun; Jackwood, Mark; Moraes, Mauro; Gardin, Yannick

2015-02-25

Vaccination is an important tool in the protection of poultry against avian influenza (AI). For field use, the overwhelming majority of AI vaccines produced are inactivated whole virus formulated into an oil emulsion. However, recombinant vectored vaccines are gaining use for their ability to induce protection against heterologous isolates and ability to overcome maternal antibody interference. In these studies, we compared protection of chickens provided by a turkey herpesvirus (HVT) vector vaccine expressing the hemagglutinin (HA) gene from a clade 2.2 H5N1 strain (A/swan/Hungary/4999/2006) against homologous H5N1 as well as heterologous H5N1 and H5N2 highly pathogenic (HP) AI challenge. The results demonstrated all vaccinated birds were protected from clinical signs of disease and mortality following homologous challenge. In addition, oral and cloacal swabs taken from challenged birds demonstrated that vaccinated birds had lower incidence and titers of viral shedding compared to sham-vaccinated birds. Following heterologous H5N1 or H5N2 HPAI challenge, 80-95% of birds receiving the HVT vector AI vaccine at day of age survived challenge with fewer birds shedding virus after challenge than sham vaccinated birds. In vitro cytotoxicity analysis demonstrated that splenic T lymphocytes from HVT-vector-AI vaccinated chickens recognized MHC-matched target cells infected with H5, as well as H6, H7, or H9 AI virus. Taken together, these studies provide support for the use of HVT vector vaccines expressing HA to protect poultry against multiple lineages of HPAI, and that both humoral and cellular immunity induced by live vaccines likely contributes to protection. Published by Elsevier Ltd.

A Novel Virus-Like Particle Based Vaccine Platform Displaying the Placental Malaria Antigen VAR2CSA.

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Susan Thrane

Full Text Available Placental malaria caused by Plasmodium falciparum is a major cause of mortality and severe morbidity. Clinical testing of a soluble protein-based vaccine containing the parasite ligand, VAR2CSA, has been initiated. VAR2CSA binds to the human receptor chondroitin sulphate A (CSA and is responsible for sequestration of Plasmodium falciparum infected erythrocytes in the placenta. It is imperative that a vaccine against malaria in pregnancy, if administered to women before they become pregnant, can induce a strong and long lasting immune response. While most soluble protein-based vaccines have failed during clinical testing, virus-like particle (VLP based vaccines (e.g., the licensed human papillomavirus vaccines have demonstrated high efficacy, suggesting that the spatial assembly of the vaccine antigen is a critical parameter for inducing an optimal long-lasting protective immune response. We have developed a VLP vaccine display platform by identifying regions of the HPV16 L1 coat protein where a biotin acceptor site (AviTagTM can be inserted without compromising VLP-assembly. Subsequent biotinylation of Avi-L1 VLPs allow us to anchor monovalent streptavidin (mSA-fused proteins to the biotin, thereby obtaining a dense and repetitive VLP-display of the vaccine antigen. The mSA-VAR2CSA antigen was delivered on the Avi-L1 VLP platform and tested in C57BL/6 mice in comparison to two soluble protein-based vaccines consisting of naked VAR2CSA and mSA-VAR2CSA. The mSA-VAR2CSA Avi-L1 VLP and soluble mSA-VAR2CSA vaccines induced higher antibody titers than the soluble naked VAR2CSA vaccine after three immunizations. The VAR2CSA Avi-L1 VLP vaccine induced statistically significantly higher endpoint titres compared to the soluble mSA-VAR2CSA vaccine, after 1st and 2nd immunization; however, this difference was not statistically significant after 3rd immunization. Importantly, the VLP-VAR2CSA induced antibodies were functional in inhibiting the binding of

Vaccine platform recombinant measles virus.

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Mühlebach, Michael D

2017-10-01

The classic development of vaccines is lengthy, tedious, and may not necessarily be successful as demonstrated by the case of HIV. This is especially a problem for emerging pathogens that are newly introduced into the human population and carry the inherent risk of pandemic spread in a naïve population. For such situations, a considerable number of different platform technologies are under development. These are also under development for pathogens, where directly derived vaccines are regarded as too complicated or even dangerous due to the induction of inefficient or unwanted immune responses causing considerable side-effects as for dengue virus. Among platform technologies are plasmid-based DNA vaccines, RNA replicons, single-round infectious vector particles, or replicating vaccine-based vectors encoding (a) critical antigen(s) of the target pathogens. Among the latter, recombinant measles viruses derived from vaccine strains have been tested. Measles vaccines are among the most effective and safest life-attenuated vaccines known. Therefore, the development of Schwarz-, Moraten-, or AIK-C-strain derived recombinant vaccines against a wide range of mostly viral, but also bacterial pathogens was quite straightforward. These vaccines generally induce powerful humoral and cellular immune responses in appropriate animal models, i.e., transgenic mice or non-human primates. Also in the recent first clinical phase I trial, the results have been quite encouraging. The trial indicated the expected safety and efficacy also in human patients, interestingly independent from the level of prevalent anti-measles immunity before the trial. Thereby, recombinant measles vaccines expressing additional antigens are a promising platform for future vaccines.

Vaccines 2.0 | Center for Cancer Research

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In 1974, Jay A. Berzofsky, M.D., Ph.D., now Chief of CCR’s Vaccine Branch, came to NIH to study protein folding. His curious mind and collaborative spirit quickly led him into the intertwined fields of immunology and vaccine development. With close to 500 publications to his name, Berzofsky has pioneered the characterization of B- and T-cell epitopes and their modification to make vaccines directed against cancer and chronic infectious diseases. He has also characterized and taken advantage of the cellular and molecular regulators of immune responses in order to enhance tumor immunity and vaccine efficacy. In the last several years, he has translated many of these strategies into promising clinical trials. From the microcosm of his laboratory, he brings the same spirit of cross-fertilizing, bench-to-bedside research to leading the Vaccine Branch as a whole.

I-MOVE multi-centre case control study 2010-11: overall and stratified estimates of influenza vaccine effectiveness in Europe.

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Esther Kissling

Full Text Available BACKGROUND: In the third season of I-MOVE (Influenza Monitoring Vaccine Effectiveness in Europe, we undertook a multicentre case-control study based on sentinel practitioner surveillance networks in eight European Union (EU member states to estimate 2010/11 influenza vaccine effectiveness (VE against medically-attended influenza-like illness (ILI laboratory-confirmed as influenza. METHODS: Using systematic sampling, practitioners swabbed ILI/ARI patients within seven days of symptom onset. We compared influenza-positive to influenza laboratory-negative patients among those meeting the EU ILI case definition. A valid vaccination corresponded to > 14 days between receiving a dose of vaccine and symptom onset. We used multiple imputation with chained equations to estimate missing values. Using logistic regression with study as fixed effect we calculated influenza VE adjusting for potential confounders. We estimated influenza VE overall, by influenza type, age group and among the target group for vaccination. RESULTS: We included 2019 cases and 2391 controls in the analysis. Adjusted VE was 52% (95% CI 30-67 overall (N = 4410, 55% (95% CI 29-72 against A(H1N1 and 50% (95% CI 14-71 against influenza B. Adjusted VE against all influenza subtypes was 66% (95% CI 15-86, 41% (95% CI -3-66 and 60% (95% CI 17-81 among those aged 0-14, 15-59 and ≥60 respectively. Among target groups for vaccination (N = 1004, VE was 56% (95% CI 34-71 overall, 59% (95% CI 32-75 against A(H1N1 and 63% (95% CI 31-81 against influenza B. CONCLUSIONS: Results suggest moderate protection from 2010-11 trivalent influenza vaccines against medically-attended ILI laboratory-confirmed as influenza across Europe. Adjusted and stratified influenza VE estimates are possible with the large sample size of this multi-centre case-control. I-MOVE shows how a network can provide precise summary VE measures across Europe.

LVIF Call 2 - Innovations to improve livestock vaccines

International Development Research Centre (IDRC) Digital Library (Canada)

Musa Mulongo

production, and commercialization of innovative vaccines against livestock ... novel vaccine design/formulations that present significant improvements over ... and which can be transferred to vaccine manufacturers for subsequent registration of.

Understanding thermostability and pH dependent properties of proteins

DEFF Research Database (Denmark)

Galberg, Pernille

The work performed in this thesis is part of a larger project (“Computational design of stable enzymes”) involving several research teams, which aimed to improve PROPKA (http://propka.ki.ku.dk) and to provide the scientific community with a computational protocol and associated PROPKA program......, which could be used for predicting mutations with expectation of increased thermostability at a certain pH value or a shifted pH activity optimum. The ability of a Bacillus circulans xylanase (BCX) mutant (N35D/A115E) to induce a decrease in pH activity optimum was evaluated by a pH dependent xylanase...

42 CFR 410.57 - Pneumococcal vaccine and flu vaccine.

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2010-10-01

... 42 Public Health 2 2010-10-01 2010-10-01 false Pneumococcal vaccine and flu vaccine. 410.57 Section 410.57 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN... § 410.57 Pneumococcal vaccine and flu vaccine. (a) Medicare Part B pays for pneumococcal vaccine and its...

Novel Vaccine Against Mycoplasma Hyosynoviae: The Immunogenic Effect of Iscom-Based Vaccines in Swine

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Lauritsen, Klara Tølbøll; Vinther Heydenreich, Annette; Riber, Ulla

Arthritis in swine is frequently caused by Mycoplasma hyosynoviae (Mhs). For the development of an effective vaccine we investigated the immunogenic effect of three vaccine preparations with the ISCOM adjuvant Posintro™ from Nordic Vaccine. A: formalin fixed whole-cells Mhs (300 µg/dose) mixed...... with Posintro, B: Deoxycholate extracted lipoproteins from Mhs organisms (DOC-antigen, 300 μg/dose) in Posintro and C: DOC-antigen (50 μg/dose) in Posintro. Each vaccine-group contained three pigs. Vaccinations (i.m.) were performed at 12 and 15 weeks of age. The development of specific IgG and secretion...... of IFNγ were measured. Three weeks after the second vaccination, pigs were euthanised and autopsied. Vaccine B induced a high level of specific serum IgG in all pigs a week after boost. Vaccine C gave a variable response after boost, with two pigs seroconverting, while no response was seen by vaccine A...

A study of vaccine-induced immune pressure on breakthrough infections in the Phambili phase 2b HIV-1 vaccine efficacy trial

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Rolland, M.; Magaret, C.A.; Rademeyer, C.; Fiore-Gartland, A.; Edlefsen, P.T.; DeCamp, A.; Ahmed, H.; Ngandu, N.; Larsen, B.B.; Frahm, N.; Marais, J.; Thebus, R.; Geraghty, D.; Hural, J.; Corey, L.; Kublin, J.; Gray, G.; McElrath, M.J.; Mullins, J.I.; Gilbert, P.B.; Williamson, C.

2016-01-01

Introduction The Merck Adenovirus-5 Gag/Pol/Nef HIV-1 subtype-B vaccine evaluated in predominately subtype B epidemic regions (Step Study), while not preventing infection, exerted vaccine-induced immune pressure on HIV-1 breakthrough infections. Here we investigated if the same vaccine exerted immune pressure when tested in the Phambili Phase 2b study in a subtype C epidemic. Materials and methods A sieve analysis, which compares breakthrough viruses from placebo and vaccine arms, was performed on 277 near full-length genomes generated from 23 vaccine and 20 placebo recipients. Vaccine coverage was estimated by computing the percentage of 9-mers that were exact matches to the vaccine insert. Results There was significantly greater protein distances from the vaccine immunogen sequence in Gag (p = 0.045) and Nef (p = 0.021) in viruses infecting vaccine recipients compared to placebo recipients. Twenty-seven putative sites of vaccine-induced pressure were identified (p sieve effect in Step was driven by HLA A*02:01; an allele which was found in low frequency in Phambili participants compared to Step participants. Furthermore, the coverage of the vaccine against subtype C Phambili viruses was 31%, 46% and 14% for Gag, Pol and Nef, respectively, compared to subtype B Step virus coverage of 56%, 61% and 26%, respectively. Discussion This study presents evidence of sieve effects in Gag and Nef; however could not confirm effects on specific amino acid sites. We propose that this weaker signal of vaccine immune pressure detected in the Phambili study compared to the Step study may have been influenced by differences in host genetics (HLA allele frequency) and reduced impact of vaccine-induced immune responses due to mismatch between the viral subtype in the vaccine and infecting subtypes. PMID:27756485

Developing Anti-HER2 Vaccines: Breast Cancer Experience.

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Al-Awadhi, Aydah; Murray, James Lee; Ibrahim, Nuhad K

2018-04-25

Breast cancer accounts for more than one million new cases annually and is the leading cause of death in women globally. HER2 overexpression induces cellular and humoral immune responses against the HER2 protein and is associated with higher tumour proliferation rates. Trastuzumab-based therapies are effectively and widely used as standard of care in HER2-amplified/overexpressed breast cancer patients; one cited mechanism of action is the induction of passive immunity and antibody-dependent cellular cytotoxicity against malignant breast cancer cells. These findings drove the efforts to generate antigen-specific immunotherapy to trigger the patient's immune system to target HER2-overexpressing tumour cells, which led to the development of various vaccines against the HER2 antigen. This manuscript discusses the various anti-HER2 vaccine formulations and strategies and their potential role in the metastatic and adjuvant settings. This article is protected by copyright. All rights reserved. © 2018 UICC.

Stimulation of mucosal immune response following oral administration of enterotoxigenic Escherichia coli fimbriae (CFA/I) entrapped in liposomes in conjunction with inactivated whole-cell Vibrio cholerae vaccine.

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Dima, V F; Ionescu, M D; Palade, R; Balotescu, C; Becheanu, G; Dima, S V

2001-01-01

In this study, we have searched for an effective mucosal vaccine. An oral enterotoxigenic E. coli vaccine containing colonization factor antigen (CFA/I) associated with inactivated whole-cell V. cholerae vaccine (WCV) has been tested for safety and immunogenicity in animals. Five groups of animals were used. The results showed the following: (a) vaccine containing CFA/I antigen entrapped in liposomes and associated with WCV (batch C) had increased titers of specific antibodies to CFA/I antigen in 15 to 18 (83.3%) animals; (b) specific Peyer's patches (PP), lymph nodes (LN) and spleen (SPL) lymphocytes proliferation was detected following in vitro restimulation with CFA/I antigen or WCV. This response gradually increased to the highest value by the 35th postimmunization day. Moreover, lower PP, LN and spleen (SPL) proliferation was observed in rabbits receiving soluble CFA/I antigen (S-CFA/I) or free liposomes (F-L) alone; (c) adhesion of E. coli H10407 strain labelled with 3H-leucine in immunized and control animals revealed the following local effects: (i) protection of rabbit intestinal mucosa against virulent E. coli cells; (ii) inhibition of adhesion of ETEC bacteria to intestinal mucosa and (iii) significantly faster release of E. coli H 10407 strain labelled with 3H-leucine from the intestinal tract of immunized animals. The histopathological and electron microscope findings confirmed the above results. The experimental results point out an efficient protection against infection with E. coli strains (ETEC), after mucosal vaccination with CFA/I antigen entrapped in liposomes associated with inactivated whole-cell Vibrio cholerae as immunological adjuvant.

Estimating the Efficacy of a Commercial Phase I Inactivated Vaccine in Decreasing the Prevalence of Coxiella burnetii Infection and Shedding in Red Deer (Cervus elaphus

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David González-Barrio

2017-12-01

Full Text Available The red deer (Cervus elaphus is a relevant reservoir for Coxiella burnetii in Iberia. C. burnetii genotypes that infect red deer also infect humans and domestic animals. Integrated control approaches that target both domestic and wild ruminants are, therefore, required to reduce C. burnetii infection risks in Iberia, especially in wildlife–livestock–human interaction scenarios. The aim of this field experiment was to test the efficacy of an inactivated phase I vaccine [Inactivated phase I vaccine (IPIV; Coxevac®] when used to control C. burnetii shedding prevalence and burden in red deer as a tool to prevent transmission to livestock and humans. A semi-extensively bred red deer population in which C. burnetii is endemic was used as a model of the Iberian context. Around 75% of the reproductive hinds (>1 year old; N = 441 in the population were first vaccinated early in 2012 and were then revaccinated 3 weeks later; they were subsequently revaccinated biannually until January 2014. 75% of the yearling females left as replacement in 2012 and 2013 were vaccinated in June and revaccinated thereafter following the same protocol. 25% of the population, including the replacement females, was kept as a control group throughout the study. Changes in the humoral immune response after vaccination were estimated by analyzing sera collected at 10 different times between January 2011 and January 2015. The vaccinated and control hinds were surveyed at 2.5, 3.5, and 4.5 months after calving in 2012, 2013, and 2014 to collect vaginal swabs, milk, and feces. The presence and burden of C. burnetii DNA in swabs, milk, and feces was evaluated by means of real-time PCR. Vaccination induced high antibody prevalence and levels. The proportion of animals shedding C. burnetii in vaginal secretions and milk did not change over time in the vaccination group with respect to the control group. In contrast, there was a significant reduction in the proportion of

Tularemia vaccine: Safety, reactogenicity, "Take" skin reactions, and antibody responses following vaccination with a new lot of the Francisella tularensis live vaccine strain - A phase 2 randomized clinical Trial.

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Mulligan, Mark J; Stapleton, Jack T; Keitel, Wendy A; Frey, Sharon E; Chen, Wilbur H; Rouphael, Nadine; Edupuganti, Srilatha; Beck, Allison; Winokur, Patricia L; El Sahly, Hana M; Patel, Shital M; Atmar, Robert L; Graham, Irene; Anderson, Edwin; El-Kamary, Samer S; Pasetti, Marcela F; Sztein, Marcelo B; Hill, Heather; Goll, Johannes B

2017-08-24

Tularemia is caused by Francisella tularensis, a gram-negative bacterium that has been weaponized as an aerosol. For protection of personnel conducting biodefense research, the United States Army required clinical evaluation of a new lot of tularemia live vaccine strain manufactured in accordance with Current Good Manufacturing Practices. A phase 2 randomized clinical trial compared the new lot (DVC-LVS) to the existing vaccine that has been in use for decades (USAMRIID-LVS). The vaccines were delivered by scarification to 228 participants. Safety, reactogenicity, take and/or antibody levels were assessed on days 0, 1, 2, 8, 14, 28, 56, and 180. Both vaccines were safe and had acceptable reactogenicity profiles during six months of follow-up. There were no serious or grade 3 and 4 laboratory adverse events. Moderate systemic reactogenicity (mostly headache or feeling tired) was reported by ∼23% of participants receiving either vaccine. Injection site reactogenicity was mostly mild itchiness and pain. The frequencies of vaccine take skin reactions were 73% (95% CI, 64, 81) for DVC-LVS and 80% (95% CI, 71, 87) for USAMRIID-LVS. The 90% CI for the difference in proportions was -6.9% (-16.4, 2.6). The rates of seroconversion measured by microagglutination assay on days 28 or 56 were 94% (95% CI, 88, 98; n=98/104) for DVC-LVS and 94% (95% CI, 87, 97; n=103/110) for USAMRIID-LVS (p=1.00). Day 14 sera revealed more rapid seroconversion for DVC-LVS relative to USAMRIID-LVS: 82% (95% CI, 73, 89) versus 55% (95% CI, 45, 65), respectively (p<0.0001). The DVC-LVS vaccine had similar safety, reactogenicity, take and antibody responses compared to the older USAMRIID vaccine, and was superior for early (day 14) antibody production. Vaccination take was not a sensitive surrogate for seroconversion in a multi-center study where personnel at five research clinics performed assessments. ClinicalTrials.gov identifier NCT01150695. Copyright © 2017 The Authors. Published by Elsevier

What you don't know about vaccines can hurt you.

Science.gov (United States)

Pace, Victor M

2015-01-01

As physicians, we've all learned in detail about the science behind vaccinations, but I suspect few of us have been taught about the history of vaccinations. Sure, we all know that Dr. Jonas Salk developed the poliovirus vaccine, but I wasn't aware that he inoculated himself, his wife, and his three children with his then experimental vaccine. When our editorial committee decided to focus on vaccinations as our theme for this month's Greene County Medical Society's Journal, I perused the internet for interesting topics. I came across a fascinating website, historyofvaccines.org; this website is a project of the College of Physicians of Philadelphia, touted as being the oldest professional medical organization in the United States. I credit the majority of the information in this article to the above website and the rest to the National Institutes of Health (nih.gov) website; I trust that the information is valid and true, based on the agencies behind these websites. Below are some interesting tidbits about vaccine preventable diseases that I found noteworthy to pass on to our readers.

Topical herpes simplex virus 2 (HSV-2) vaccination with human papillomavirus vectors expressing gB/gD ectodomains induces genital-tissue-resident memory CD8+ T cells and reduces genital disease and viral shedding after HSV-2 challenge.

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Çuburu, Nicolas; Wang, Kening; Goodman, Kyle N; Pang, Yuk Ying; Thompson, Cynthia D; Lowy, Douglas R; Cohen, Jeffrey I; Schiller, John T

2015-01-01

No herpes simplex virus 2 (HSV-2) vaccine has been licensed for use in humans. HSV-2 glycoproteins B (gB) and D (gD) are targets of neutralizing antibodies and T cells, but clinical trials involving intramuscular (i.m.) injection of HSV-2 gB and gD in adjuvants have not been effective. Here we evaluated intravaginal (ivag) genetic immunization of C57BL/6 mice with a replication-defective human papillomavirus pseudovirus (HPV PsV) expressing HSV-2 gB (HPV-gB) or gD (HPV-gD) constructs to target different subcellular compartments. HPV PsV expressing a secreted ectodomain of gB (gBsec) or gD (gDsec), but not PsV expressing a cytoplasmic or membrane-bound form, induced circulating and intravaginal-tissue-resident memory CD8(+) T cells that were able to secrete gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) as well as moderate levels of serum HSV neutralizing antibodies. Combined immunization with HPV-gBsec and HPV-gDsec (HPV-gBsec/gDsec) vaccines conferred longer survival after vaginal challenge with HSV-2 than immunization with HPV-gBsec or HPV-gDsec alone. HPV-gBsec/gDsec ivag vaccination was associated with a reduced severity of genital lesions and lower levels of viral shedding in the genital tract after HSV-2 challenge. In contrast, intramuscular vaccination with a soluble truncated gD protein (gD2t) in alum and monophosphoryl lipid A (MPL) elicited high neutralizing antibody titers and improved survival but did not reduce genital lesions and viral shedding. Vaccination combining ivag HPV-gBsec/gDsec and i.m. gD2t-alum-MPL improved survival and reduced genital lesions and viral shedding. Finally, high levels of circulating HSV-2-specific CD8(+) T cells, but not serum antibodies, correlated with reduced viral shedding. Taken together, our data underscore the potential of HPV PsV as a platform for a topical mucosal vaccine to control local manifestations of primary HSV-2 infection. Genital herpes is a highly prevalent chronic disease caused by

Characterization of a cold-active esterase from Serratia sp. and improvement of thermostability by directed evolution.

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Jiang, Huang; Zhang, Shaowei; Gao, Haofeng; Hu, Nan

2016-01-22

In recent years, cold-active esterases have received increased attention due to their attractive properties for some industrial applications such as high catalytic activity at low temperatures. An esterase-encoding gene (estS, 909 bp) from Serratia sp. was identified, cloned and expressed in Escherichia coli DE3 (BL21). The estS encoded a protein (EstS) of 302 amino acids with a predicted molecular weight of 32.5 kDa. It showed the highest activity at 10 °C and pH 8.5. EstS was cold active and retained ~92 % of its original activity at 0 °C. Thermal inactivation analysis showed that the T1/2 value of EstS was 50 min at 50 °C (residual activity 41.23 %) after 1 h incubation. EstS is also quite stable in high salt conditions and displayed better catalytic activity in the presence of 4 M NaCl. To improve the thermo-stability of EstS, variants of estS gene were created by error-prone PCR. A mutant 1-D5 (A43V, R116W, D147N) that showed higher thermo-stability than its wild type predecessor was selected. 1-D5 showed enhanced T1/2 of 70 min at 50 °C and retained 63.29 % of activity after incubation at 50 °C for 60 min, which were about 22 % higher than the wild type (WT). CD spectrum showed that the secondary structure of WT and 1-D5 are more or less similar, but an increase in β-sheets was recorded, which enhanced the thermostability of mutant protein. EstS was a novel cold-active and salt-tolerant esterase and half-life of mutant 1-D5 was enhanced by 1.4 times compared with WT. The features of EstS are interesting and can be exploited for commercial applications. The results have also provided useful information about the structure and function of Est protein.

Low rate of human papillomavirus vaccination among schoolgirls in Lebanon: barriers to vaccination with a focus on mothers’ knowledge about available vaccines

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Abou El-Ola MJ

2018-03-01

Full Text Available Maria J Abou El-Ola,1 Mariam A Rajab,2 Dania I Abdallah,3 Ismail A Fawaz,4 Lyn S Awad,5 Hani M Tamim,6 Ahmad O Ibrahim,7 Anas M Mugharbil,7 Rima A Moghnieh8 1Department of Obstetrics and Gynaecology, Makassed General Hospital, Beirut, Lebanon; 2Department of Pediatrics, Makassed General Hospital, Beirut, Lebanon; 3Department of Pharmacy, Makassed General Hospital, Beirut, Lebanon; 4Department of Internal Medicine, Iklim Health Foundation Hospital, Mazboud, Mount Lebanon, Chouf, Lebanon; 5Department of Pharmacy, Makassed General Hospital, Beirut, Lebanon; 6Department of Internal Medicine, American University of Beirut, Beirut, Lebanon; 7Division of Hematology/Oncology, Department of Internal Medicine, Makassed General Hospital, Beirut, Lebanon; 8Division of Infectious Diseases, Department of Internal Medicine, Makassed General Hospital, Beirut, Lebanon Background: Human papillomavirus (HPV infection is an established predisposing factor of cervical cancer. In this study, we assessed the awareness about genital warts, cervical cancer, and HPV vaccine among mothers having girls who are at the age of primary HPV vaccination attending a group of schools in Lebanon. We also assessed the rate of HPV vaccination among these girls and the barriers to vaccination in this community. Subjects and methods: This is a cross-sectional, school-based survey. A 23-item, self-administered, anonymous, pretested, structured questionnaire with closed-ended questions was used to obtain data. The questionnaire was sent to the mothers through their student girls, and they were asked to return it within a week. Data were analyzed using the Statistical Package for Social Sciences version 21.0. Bivariate analysis was performed using the chi-square test to compare categorical variables, whereas continuous variables were compared using the Student’s t-test. Fisher’s exact test was used when chi-square test could not be employed. Results: The response rate in our survey

Motivations to participate in a Phase I/II HIV vaccine trial: A descriptive study from Dar es Salaam, Tanzania

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E. A. M. Tarimo

2016-02-01

Full Text Available Abstract Background The search for an efficacious HIV vaccine is a global priority. To date only one HIV vaccine trial (RV144 has shown modest efficacy in a phase III trial. With existing different HIV-1 subtypes and frequent mutations, multiple trials are needed from different geographical sites particularly in sub-Saharan Africa where most HIV infections occur. Thus, motivations to participate in HIV vaccine trials among Tanzanians need to be assessed. This paper describes the motives of Police Officers who showed great interest to volunteer in HIVIS-03 in Dar es Salaam, Tanzania. Methods A descriptive cross-sectional study was conducted among Police Officers who showed interest to participate in the HIVIS-03, a phase I/II HIV vaccine trial in Dar es Salaam. Prior to detailed training sessions about HIV vaccine trials, the potential participants narrated their individual motives to participate in the trial on a piece of paper. Descriptive analysis using content approach and frequency distributions were performed. Results Of the 265 respondents, 242 (91.3 % provided their socio-demographic characteristics as well as reasons that would make them take part in the proposed trial. Majority, (39.7 %, cited altruism as the main motive. Women were more likely to volunteer due to altruism compared to men (P < 0.01. Researchers’ explanations about HIV/AIDS vaccine studies motivated 15.3 %. More men (19.6 % than women (1.7 % were motivated to volunteer due to researchers’ explanations (P < 0.001. Also, compared to other groups, those unmarried and educated up to secondary level of education were motivated to volunteer due to researchers’ explanation (P < 0.05. Other reasons were: desire to become a role model (18.6 %; to get knowledge for educating others (14.0 %; to cooperate with researchers in developing an HIV vaccine (9.5 %; to get protection against HIV infection (7.0 %, and severity of the disease within families (6.2�

Optimization of physical factors affecting the production of thermo-stable organic solvent-tolerant protease from a newly isolated halo tolerant Bacillus subtilis strain Rand

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Salleh Abu

2009-04-01

Full Text Available Abstract Background Many researchers have reported on the optimization of protease production; nevertheless, only a few have reported on the optimization of the production of organic solvent-tolerant proteases. Ironically, none has reported on thermostable organic solvent-tolerant protease to date. The aim of this study was to isolate the thermostable organic solvent-tolerant protease and identify the culture conditions which support its production. The bacteria of genus Bacillus are active producers of extra-cellular proteases, and the thermostability of enzyme production by Bacillus species has been well-studied by a number of researchers. In the present study, the Bacillus subtilis strain Rand was isolated from the contaminated soil found in Port Dickson, Malaysia. Results A thermostable organic solvent-tolerant protease producer had been identified as Bacillus subtilis strain Rand, based on the 16S rRNA analysis conducted, as well as the morphological characteristics and biochemical properties. The production of the thermostable organic solvent-tolerant protease was optimized by varying various physical culture conditions. Inoculation with 5.0% (v/v of (AB600 = 0.5 inoculum size, in a culture medium (pH 7.0 and incubated for 24 h at 37°C with 200 rpm shaking, was the best culture condition which resulted in the maximum growth and production of protease (444.7 U/ml; 4042.4 U/mg. The Rand protease was not only stable in the presence of organic solvents, but it also exhibited a higher activity than in the absence of organic solvent, except for pyridine which inhibited the protease activity. The enzyme retained 100, 99 and 80% of its initial activity, after the heat treatment for 30 min at 50, 55, and 60°C, respectively. Conclusion Strain Rand has been found to be able to secrete extra-cellular thermostable organic solvent-tolerant protease into the culture medium. The protease exhibited a remarkable stability towards temperature and organic

Efficacy and safety of a new intradermal PCV2 vaccine in pigs

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M. Sno

2016-01-01

Vaccination with Porcilis® PCV ID resulted in small transient local reactions in a high percentage of the vaccinated animals with no temperature increase. In both the onset of immunity and duration of immunity challenge studies with PCV2 or M. hyopneumoniae, significant reduction of the PCV2 load in lymphoid tissue, lungs, serum and fecal swabs and M. hyopneumoniae-induced lung lesions were observed. In two field trials on two different farms where both PCV2 and M. hyopneumoniae were present, vaccination with Porcilis® PCV ID and/or Porcilis® M Hyo ID ONCE of 3 week old piglets resulted in a significant reduction of PCV2 viraemia, mortality and lung lesion scores at slaughter. In addition, a significant positive effect on average daily weight gain (between 44 and 59 g/day in the finishing phase was observed. The results support that this new intradermal vaccine is safe and efficacious against PCV2 and may be used concurrently with Porcilis® M Hyo ID ONCE.

Essays on the collective action dilemma of vaccination

OpenAIRE

Ahlskog, Rafael

2017-01-01

Vaccines famously possess positive externalities that make them susceptible to the collective action dilemma: when I get vaccinated, I protect not only myself, but also those who I might otherwise have infected. Thus, some people will have an incentive to free ride on the immunity of others. In a population of rational agents, the critical level of vaccination uptake required for herd immunity will therefore be difficult to attain in the long run, which poses difficulties for disease eradicat...

Population-level effect of potential HSV2 prophylactic vaccines on HIV incidence in sub-Saharan Africa

Science.gov (United States)

Freeman, Esther E.; White, Richard G.; Bakker, Roel; Orroth, Kate K.; Weiss, Helen A.; Buvé, Anne; Hayes, Richard J.; Glynn, Judith R.

2009-01-01

Herpes simplex virus type-2 (HSV2) infection increases HIV transmission. We explore the impact of a potential prophylactic HSV2 vaccination on HIV incidence in Africa using STDSIM an individual-based model. A campaign that achieved 70% coverage over 5 years with a vaccine that reduced susceptibility to HSV2 acquisition and HSV2 reactivation by 75% for 10 years, reduced HIV incidence by 30–40% after 20 years (range 4–66%). Over 20 years, in most scenarios fewer than 100 vaccinations were required to avert one HIV infection. HSV2 vaccines could have a substantial impact on HIV incidence. Intensified efforts are needed to develop an effective HSV2 vaccine. PMID:19071187

Studies on Lyophilization of Sabin Vaccine. 2. Investigation on Long Time Incubation at High and Low Temperatures of Lyophilized Sabin Vaccine

OpenAIRE

塩見, 洋; 浦沢, 价子; 浦沢, 正三

1998-01-01

Lyophilization of vaccine is one of the possible options for the development of a heat-stable poliovaccine. In our previous study in which conditions affecting the lyophilization of Sabin poliovaccine were investigated, it was found that infectivity titration of lyophilized viruses was under at least three kind of variabilities i. e., i) the variability among different lyophilization experi-ments, ii) the variability within the same lyophilization experiment and iii) the variability inherent ...

A novel engineered interchain disulfide bond in the constant region enhances the thermostability of adalimumab Fab.

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Nakamura, Hitomi; Oda-Ueda, Naoko; Ueda, Tadashi; Ohkuri, Takatoshi

2018-01-01

We constructed a system for expressing the Fab of the therapeutic human monoclonal antibody adalimumab at a yield of 20 mg/L in the methylotrophic yeast Pichia pastoris. To examine the contribution of interchain disulfide bonds to conformational stability, we prepared adalimumab Fab from which the interchain disulfide bond at the C-terminal region at both the CH 1 and CL domains was deleted by substitution of Cys with Ala (Fab ΔSS ). DSC measurements showed that the Tm values of Fab ΔSS were approximately 5 °C lower than those of wild-type Fab, suggesting that the interchain disulfide bond contributes to conformational thermostability. Using computer simulations, we designed a novel interchain disulfide bond outside the C-terminal region to increase the stability of Fab ΔSS . The resulting Fab (mutSS Fab ΔSS ) had the mutations H:V177C and L:Q160C in Fab ΔSS , confirming the formation of the disulfide bond between CH 1 and CL. The thermostability of mutSS Fab ΔSS was approximately 5 °C higher than that of Fab ΔSS . Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of Fab ΔSS and mitigated the destabilization caused by partial reduction of the interchain disulfide bond at the C-terminal region, which occurs in site-specific modification such as PEGylation. Copyright © 2017 Elsevier Inc. All rights reserved.

Clinical cancer chemoprevention: From the hepatitis B virus (HBV vaccine to the human papillomavirus (HPV vaccine

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Horng-Jyh Tsai

2015-04-01

Full Text Available Approximately 2 million new cancer cases are attributed to infectious agents each year worldwide. Vaccines for the hepatitis B virus (HBV, a risk factor of hepatocellular cancer, and human papillomavirus (HPV, a risk factor of cervical cancer, are considered major successes in clinical chemoprevention of cancer. In Taiwan, the first evidence of cancer prevention through vaccinations was provided by HBV vaccination data in infants. The Taiwanese HBV vaccination program has since become a model immunization schedule for newborns worldwide. Persistent infection with high-risk HPV is generally accepted as prerequisite for cervical cancer diagnosis; however, cervical cancer is a rare complication of HPV infections. This is due to the fact that such infections tend to be transient. The safety and efficacy of both available HPV quadrivalent vaccine and bivalent vaccine are not in doubt at the present time. Until a human cytomegalovirus (CMV vaccine becomes available, simple hygienic practices, such as hand washing, can prevent CMV infection both before and during pregnancy. Each country should establish her official guidelines regarding which vaccines should be used to treat various conditions, the target population (i.e., universal or limited to a selected population, and the immunization schedules. After a vaccine is recommended, decisions regarding reimbursement by the public health care fund are evaluated. The guidelines become part of the immunization schedule, which is updated annually and published in the official bulletin. In conclusion, both HBV and HPV vaccines are considered major successes in the chemoprevention of cancer.

Comparative study on immuno-modulatory effect of anticoccidial vaccines and a coccidiostat on nd vaccinated broiler chicks

International Nuclear Information System (INIS)

Hazeen, M.K.; Mustafa, M.Y.; But, T.M.

2007-01-01

105 day old broiler chicks were divided into 5 equal groups with 21 chicks in each i.e. A (non vaccinated, non medicated control), B (ND vaccinated control), C (administered with ND vaccine Lasota and Eimeria vaccine EV), D (NDV and immucox) and E (administered with NDV vaccine and coccidiostat). The comparative immunomodulatory effects of EV, immucox and Sacox were worked out on the basis of the GMT levels in Newcastle disease vaccinated birds along with the non-vaccinated and non-medicated control birds. These titres were evaluated by using HA and H1 tests on the sera of these experimental chicks. Other parameters i.e. morbidity, mortality, OPG, weight gain, FCR, Postmortem findings, Bursal/body weight ratio, weight, size and texture of spleen, thymus and liver were also assessed. The birds that were kept as NDV vaccinated control (group B) had the highest body weight and showed best FCR. The birds of unmedicated, unvaccinated control group (A) secured 2nd position regarding weight gain and FCR. Among the three experimental groups (C, D and E), the birds from group C (NDV + EV) had higher body weight than group D and E. Feed conversion ratio (FCR) of the birds of group C was also found to be better than the birds belonging to group D and E. The number of oocysts per gm of faeces showed gradual decrease and became nil on the observation of day 33 of the experiment, as the birds became solidly immune against Eimeria infection at this age. The birds belonging to group B (NDV vaccinated control) had shown the highest antibody titers while the birds of group A (unvaccinated unmedicated control) had the lowest antibody titres. Among the three experimental groups (C, D and E) the birds from group C had the higher antibody titres as compared to other two groups (D and E) on day 49. (author)

MOLECULAR CLONING AND CHARACTERIZATION OF NOVEL THERMOSTABLE LIPASE FROM SHEWANELLA PUTREFACIENS AND USING ENZYMATIC BIODIESEL PRODUCTION

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Fahri Akbas

2015-02-01

Full Text Available A novel thermostable lipase from Shewanella putrefaciens was identified, expressed in Escherichia coli, characterized and used in biodiesel production. Enzyme characterization was carried out by enzyme assay, SDS-PAGE and other biochemical reactions. The recombinant lipase was found to have a molecular mass of 29 kDa and exhibited lipase activity when Tween 80 was used as the substrate. The purified enzyme showed maximum activity at pH 5.0 and at 80°C. The recombinant lipase was used for the transesterification of canola oil and waste oil. The enzyme retains 50% of its activity at 90°C for 30 minutes. It is also able to retain 20% of its activity even at 100 °C for 20 minutes. These properties of the obtained new recombinant thermostable lipase make it promising as a biocatalyst for industrial processes.

Novel multi-peptide vaccination in Hla-A2+ hormone sensitive patients with biochemical relapse of prostate cancer.

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Feyerabend, Susan; Stevanovic, Stefan; Gouttefangeas, Cécile; Wernet, Dorothee; Hennenlotter, Jörg; Bedke, Jens; Dietz, Klaus; Pascolo, Steve; Kuczyk, Markus; Rammensee, Hans-Georg; Stenzl, Arnulf

2009-06-15

A phase I/II trial was conducted to assess feasibility and tolerability of tumor associated antigen peptide vaccination in hormone sensitive prostate carcinoma (PC) patients with biochemical recurrence after primary surgical treatment. Nineteen HLA-A2 positive patients with rising PSA without detectable metastatic disease or local recurrence received 11 HLA-A*0201-restricted and two HLA class II synthetic peptides derived from PC tumor antigens subcutaneously for 18 months or until PSA progression. The vaccine was emulgated in montanide ISA51 and combined with imiquimod, GM-CSF, mucin-1-mRNA/protamine complex, local hyperthermia or no adjuvant. PSA was assessed, geometric mean doubling times (DT) calculated and clinical performance monitored. PSA DT of 4 out of 19 patients (21%) increased from 4.9 to 25.8 months during vaccination. Out of these, two patients (11%) exhibited PSA stability for 28 and 31 months which were still continuing at data cut-off. One patient showed no change of PSA DT during vaccination but decline after the therapy. Three patients had an interim PSA decline or DT increase followed by DT decrease compared to baseline PSA DT. Three of the responding patients received imiquimod and one the mucin-1-mRNA/protamine complex as adjuvant; both are Toll-like receptor-7 agonists. Eleven (58%) patients had progressive PSA values. The vaccine was well tolerated, and no grade III or IV toxicity occurred. Multi-peptide vaccination stabilized or slowed down PSA progress in four of 19 cases. The vaccination approach is promising with moderate adverse events. Long-term stability delayed androgen deprivation up to 31 months. TLR-7 co-activation seems to be beneficial.

Influence of porcine circovirus type 2 vaccination on the probability and severity of pneumonia detected postmortem.

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Raith, J; Kuchling, S; Schleicher, C; Schobesberger, H; Köfer, J

2015-01-31

To evaluate the influence of porcine circovirus type 2 vaccination (PCV-2) on the probability and severity of pneumonia, postmortem findings of 247,505 pigs slaughtered between 2008 and 2011 were analysed by applying a cumulative link mixed model. Three major effects could be observed: (1) PCV-2 vaccination significantly (P<0.01) reduced the odds (coefficient: -0.05) of postmortem findings of mild, moderate and severe pneumonia for vaccinated pigs. (2) Pigs from fattening farms were less likely (coefficient: -0.44; P<0.05) to exhibit signs of pneumonia at slaughter than pigs from farrow-to-finish farms. (3) When vaccinated, the odds of detecting postmortem signs showed an even more pronounced reduction (coefficient: -0.19; P<0.001) for pigs from fattening farms. Combining PCV-2 vaccination, farm type and interaction effects between these two factors, a pig vaccinated against PCV-2 from a fattening farm had only half the chance (OR 0.51) of pneumonia being detected at postmortem than a non-vaccinated pig from a farrow-to-finish farm. The study demonstrates the benefit of a vaccination programme against PCV-2 as an important tool to reduce the risk of postmortem pneumonia findings and the severity of pneumonia in pigs at slaughter. British Veterinary Association.

Attenuated Salmonella enterica serovar Typhi and Shigella flexneri 2a strains mucosally deliver DNA vaccines encoding measles virus hemagglutinin, inducing specific immune responses and protection in cotton rats.

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Pasetti, Marcela F; Barry, Eileen M; Losonsky, Genevieve; Singh, Mahender; Medina-Moreno, Sandra M; Polo, John M; Ulmer, Jeffrey; Robinson, Harriet; Sztein, Marcelo B; Levine, Myron M

2003-05-01

Measles remains a leading cause of child mortality in developing countries. Residual maternal measles antibodies and immunologic immaturity dampen immunogenicity of the current vaccine in young infants. Because cotton rat respiratory tract is susceptible to measles virus (MV) replication after intranasal (i.n.) challenge, this model can be used to assess the efficacy of MV vaccines. Pursuing a new measles vaccine strategy that might be effective in young infants, we used attenuated Salmonella enterica serovar Typhi CVD 908-htrA and Shigella flexneri 2a CVD 1208 vaccines to deliver mucosally to cotton rats eukaryotic expression plasmid pGA3-mH and Sindbis virus-based DNA replicon pMSIN-H encoding MV hemagglutinin (H). The initial i.n. dose-response with bacterial vectors alone identified a well-tolerated dosage (1 x 10(9) to 7 x 10(9) CFU) and a volume (20 micro l) that elicited strong antivector immune responses. Animals immunized i.n. on days 0, 28, and 76 with bacterial vectors carrying DNA plasmids encoding MV H or immunized parenterally with these naked DNA vaccine plasmids developed MV plaque reduction neutralizing antibodies and proliferative responses against MV antigens. In a subsequent experiment of identical design, cotton rats were challenged with wild-type MV 1 month after the third dose of vaccine or placebo. MV titers were significantly reduced in lung tissue of animals immunized with MV DNA vaccines delivered either via bacterial live vectors or parenterally. Since attenuated serovar Typhi and S. flexneri can deliver measles DNA vaccines mucosally in cotton rats, inducing measles immune responses (including neutralizing antibodies) and protection, boosting strategies can now be evaluated in animals primed with MV DNA vaccines.

Tdap (tetanus, diphtheria, pertussis) Vaccine and Pregnancy

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... Can I receive the Tdap vaccine while breastfeeding? Yes. Noninfectious vaccines like Tdap are compatible with breastfeeding. If you get the vaccine while breastfeeding, it can help prevent you from getting sick and passing the illness to your baby. ...

Timeliness vaccination of measles containing vaccine and barriers to vaccination among migrant children in East China.

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Yu Hu

Full Text Available BACKGROUND: The reported coverage rates of first and second doses of measles containing vaccine (MCV are almost 95% in China, while measles cases are constantly being reported. This study evaluated the vaccine coverage, timeliness, and barriers to immunization of MCV1 and MCV2 in children aged from 8-48 months. METHODS: We assessed 718 children aged 8-48 months, of which 499 children aged 18-48 months in September 2011. Face to face interviews were administered with children's mothers to estimate MCV1 and MCV2 coverage rate, its timeliness and barriers to vaccine uptake. RESULTS: The coverage rates were 76.9% for MCV1 and 44.7% for MCV2 in average. Only 47.5% of surveyed children received the MCV1 timely, which postpone vaccination by up to one month beyond the stipulated age of 8 months. Even if coverage thus improves with time, postponed vaccination adds to the pool of unprotected children in the population. Being unaware of the necessity for vaccination and its schedule, misunderstanding of side-effect of vaccine, and child being sick during the recommended vaccination period were significant preventive factors for both MCV1 and MCV2 vaccination. Having multiple children, mother's education level, household income and children with working mothers were significantly associated with delayed or missing MCV1 immunization. CONCLUSIONS: To avoid future outbreaks, it is crucial to attain high coverage levels by timely vaccination, thus, accurate information should be delivered and a systematic approach should be targeted to high-risk groups.

DNA fusion gene vaccines

DEFF Research Database (Denmark)

Holst, Peter Johannes; Bassi, Maria Rosaria; Thomsen, Allan Randrup

2010-01-01

DNA vaccines are versatile and safe, but limited immunogenicity has prevented their use in the clinical setting. Experimentally, immunogenicity may be enhanced by the use of new delivery technologies, by coadministration of cytokines and pathogen-associated molecular patterns, or by fusion...... of antigens into molecular domains that enhance antigen presentation. More specifically, the immunogenicity of DNA vaccines may benefit from increased protein synthesis, increased T-cell help and MHC class I presentation, and the addition of a range of specific cytokines and pathogen-associated molecular...... with viral-vectored vaccines, various synergistic components may need to be incorporated into DNA vaccines. From the perspective of the future clinical use of DNA vaccines, it has been suggested that antigen presentation should be improved and cytokine coadministration attempted. However, even...

Cost-effectiveness of human papillomavirus vaccination and cervical cancer screening in Thailand.

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Sharma, M; Ortendahl, J; van der Ham, E; Sy, S; Kim, J J

2012-01-01

To assess the health and economic outcomes of various screening and vaccination strategies for cervical cancer prevention. Cost-effectiveness analysis from a societal perspective. Thailand. Females aged 9 years and older. Using a mathematical model of human papillomavirus (HPV) infection and cervical cancer, calibrated to epidemiological data from Thailand, we estimated the cost-effectiveness of pre-adolescent HPV vaccination, screening [visual inspection with acetic acid (VIA), HPV DNA testing, and cytology] between one and five times per lifetime in adulthood, and combined pre-adolescent vaccination and screening. Vaccine efficacy, coverage, cost, and screening frequency were varied in sensitivity analyses. Incremental cost-effectiveness ratios, expressed as cost per year of life saved (YLS). Assuming lifelong efficacy and 80% coverage, pre-adolescent HPV vaccination alone was projected to reduce the lifetime risk of cervical cancer by 55%, which was greater than any strategy of screening alone. When cost per vaccinated girl was I$10 (approximately $2 per dose) or less, HPV vaccination alone was cost saving. Pre-adolescent vaccination and HPV DNA testing five times per lifetime, starting at age 35 years, reduced the lifetime cervical cancer risk by 70%, and had a cost-effectiveness ratio less than Thailand's GDP per capita (I$8100), provided the cost per vaccinated girl was I$200 or less. Low cost pre-adolescent HPV vaccination followed by HPV screening five times per lifetime is an efficient strategy for Thailand. Costs may need to be lower, however, for this strategy to be affordable. If vaccination is not feasible, HPV DNA testing five times per lifetime is efficient. © 2011 The Authors BJOG An International Journal of Obstetrics and Gynaecology © 2011 RCOG.

Determination of the minimum protective dose for bluetongue virus serotype 2 and 8 vaccines in sheep.

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Modumo, Jacob; Venter, Estelle H

2012-08-03

Recent outbreaks of bluetongue virus (BTV) serotypes 2 and 8 in many European countries provided an opportunity to investigate the possibility of improving the safety of the modified live vaccines administered mainly in South Africa. Modified live vaccines (MLV) released at a titre of 5 x 104 PFU/mL, raised concerns and prompted the need to determine the minimum titre which will still be protective and also safe. The BTV serotypes 2 and 8 vaccines were produced at the following titres: 102 PFU/mL, 103 PFU/mL and 104 PFU/mL, and were injected into 24 sheep which were then monitored. Blood was collected on days 0, 3, 6, 9, 12, 15, 18, 21, 25, 28 and 4 months post vaccination, for seroconversion and viraemia studies. These sheep were later challenged at 4 months post vaccination using BTV infected cell culture material, they were then observed and bled and again tested for viraemia. There was no viraemia post vaccination, however, a febrile reaction did occur and seroconversion was demonstrated at low titres for both BTV 2 and 8. Although viraemia was demonstrated post challenge, sheep vaccinated with the low titre BTV 2 vaccine showed more than a 90% protection index at a lower titre of 103 PFU/mL, compared with BTV 8 that showed a protection index above 90% at all the titres used. It is recommended that for BTV 2 vaccine, sheep should be vaccinated at a titre of 103 PFU/mL and at a titre of 102 PFU/mL with BTV 8 vaccine.

Phase I safety and immunogenicity evaluation of MVA-CMDR, a multigenic, recombinant modified vaccinia Ankara-HIV-1 vaccine candidate.

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Currier, Jeffrey R; Ngauy, Viseth; de Souza, Mark S; Ratto-Kim, Silvia; Cox, Josephine H; Polonis, Victoria R; Earl, Patricia; Moss, Bernard; Peel, Sheila; Slike, Bonnie; Sriplienchan, Somchai; Thongcharoen, Prasert; Paris, Robert M; Robb, Merlin L; Kim, Jerome; Michael, Nelson L; Marovich, Mary A

2010-11-15

We conducted a Phase I randomized, dose-escalation, route-comparison trial of MVA-CMDR, a candidate HIV-1 vaccine based on a recombinant modified vaccinia Ankara viral vector expressing HIV-1 genes env/gag/pol. The HIV sequences were derived from circulating recombinant form CRF01_AE, which predominates in Thailand. The objective was to evaluate safety and immunogenicity of MVA-CMDR in human volunteers in the US and Thailand. MVA-CMDR or placebo was administered intra-muscularly (IM; 10(7) or 10(8) pfu) or intradermally (ID; 10(6) or 10(7) pfu) at months 0, 1 and 3, to 48 healthy volunteers at low risk for HIV-1 infection. Twelve volunteers in each dosage group were randomized to receive MVA-CMDR or placebo (10∶2). Volunteers were actively monitored for local and systemic reactogenicity and adverse events post vaccination. Cellular immunogenicity was assessed by a validated IFNγ Elispot assay, an intracellular cytokine staining assay, lymphocyte proliferation and a (51)Cr-release assay. Humoral immunogenicity was assessed by ADCC for gp120 and binding antibody ELISAs for gp120 and p24. MVA-CMDR was safe and well tolerated with no vaccine related serious adverse events. Cell-mediated immune responses were: (i) moderate in magnitude (median IFNγ Elispot of 78 SFC/10(6) PBMC at 10(8) pfu IM), but high in response rate (70% (51)Cr-release positive; 90% Elispot positive; 100% ICS positive, at 10(8) pfu IM); (ii) predominantly HIV Env-specific CD4(+) T cells, with a high proliferative capacity and durable for at least 6 months (100% LPA response rate by the IM route); (iv) dose- and route-dependent with 10(8) pfu IM being the most immunogenic treatment. Binding antibodies against gp120 and p24 were detectable in all vaccination groups with ADCC capacity detectable at the highest dose (40% positive at 10(8) pfu IM). MVA-CMDR delivered both intramuscularly and intradermally was safe, well-tolerated and elicited durable cell-mediated and humoral immune responses

Phase I safety and immunogenicity evaluation of MVA-CMDR, a multigenic, recombinant modified vaccinia Ankara-HIV-1 vaccine candidate.

Directory of Open Access Journals (Sweden)

Jeffrey R Currier

2010-11-01

Full Text Available We conducted a Phase I randomized, dose-escalation, route-comparison trial of MVA-CMDR, a candidate HIV-1 vaccine based on a recombinant modified vaccinia Ankara viral vector expressing HIV-1 genes env/gag/pol. The HIV sequences were derived from circulating recombinant form CRF01_AE, which predominates in Thailand. The objective was to evaluate safety and immunogenicity of MVA-CMDR in human volunteers in the US and Thailand.MVA-CMDR or placebo was administered intra-muscularly (IM; 10(7 or 10(8 pfu or intradermally (ID; 10(6 or 10(7 pfu at months 0, 1 and 3, to 48 healthy volunteers at low risk for HIV-1 infection. Twelve volunteers in each dosage group were randomized to receive MVA-CMDR or placebo (10∶2. Volunteers were actively monitored for local and systemic reactogenicity and adverse events post vaccination. Cellular immunogenicity was assessed by a validated IFNγ Elispot assay, an intracellular cytokine staining assay, lymphocyte proliferation and a (51Cr-release assay. Humoral immunogenicity was assessed by ADCC for gp120 and binding antibody ELISAs for gp120 and p24. MVA-CMDR was safe and well tolerated with no vaccine related serious adverse events. Cell-mediated immune responses were: (i moderate in magnitude (median IFNγ Elispot of 78 SFC/10(6 PBMC at 10(8 pfu IM, but high in response rate (70% (51Cr-release positive; 90% Elispot positive; 100% ICS positive, at 10(8 pfu IM; (ii predominantly HIV Env-specific CD4(+ T cells, with a high proliferative capacity and durable for at least 6 months (100% LPA response rate by the IM route; (iv dose- and route-dependent with 10(8 pfu IM being the most immunogenic treatment. Binding antibodies against gp120 and p24 were detectable in all vaccination groups with ADCC capacity detectable at the highest dose (40% positive at 10(8 pfu IM.MVA-CMDR delivered both intramuscularly and intradermally was safe, well-tolerated and elicited durable cell-mediated and humoral immune responses

Neonate exposure to thimerosal mercury from hepatitis B vaccines.

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Dórea, José G; Marques, Rejane C; Brandão, Katiane G

2009-08-01

Infant exposure to ethylmercury (EtHg) has not only increased but is starting earlier as a result of the current immunization schedule that uses thimerosal-containing vaccines (TCVs). Although vaccination schedule varies considerably between countries, infants in less-developed countries continue to be exposed to EtHg derived from more affordable TCVs. We studied the exposure of newborns to EtHg from hepatitis B vaccines; hospital records (21,685) were summarized for the years 2001 to 2005 regarding date of birth, vaccination date, and birth weight. Most of the vaccinations occurred in the first 24 hours postdelivery; over the 5 years, there was an increase in vaccinations within hours of birth (same day), from 7.4% (2001) to 87.8% (2005). Nearly 94.6% of infants are now being vaccinated within the first 24 hours. Range of mercury exposure spread from 4.2 to 21.1 microg mercury/kg body weight for those receiving TCVs with the highest thimerosal concentration; these exposure levels are conservative for 2% of children receiving vaccines within 2 to 3 postnatal days, when they are still going through physiological postnatal weight loss. Because of the particular timing (transitioning from in utero to ex utero metabolism) and specific aspects of exposure (i.e., parenteral mode, bypassing gastroenteric barriers) and dose (related to vaccine manufacturer and with variation in birth weight), this study reveals critical issues that can modulate toxicokinetics and toxicodynamics of organomercurials in neonates.

Progression of rabbit haemorrhagic disease virus 2 upon vaccination in an industrial rabbitry: a laboratorial approach

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C.L. Carvalho

2017-03-01

Full Text Available Rabbit haemorrhagic disease virus 2 (RHDV2 emerged recently in several European countries, leading to extensive economic losses in the industry. In response to this new infection, specific inactivated vaccines were developed in Europe and full and rapid setup of protective immunity induced by vaccination was reported. However, data on the efficacy of these vaccines in an ongoing-infection scenario is unavailable. In this study we investigated an infected RHDV2 indoor industrial meat rabbitry, where fatalities continued to occur after the implementation of the RHDV2 vaccination, introduced to control the disease. The aim of this study was to understand if these mortalities were RHDV2-related, to discover if the dead animals showed any common features such as age or time distance from vaccination, and to identify the source of the outbreak. Anatomo-pathological analysis of vaccinated animals with the virus showed lesions compatible with systemic haemorrhagic disease and RHDV2-RNA was detected in 85.7% of the animals tested. Sequencing of the vp60 gene amplified from liver samples led to the recognition of RHDV2 field strains demonstrating that after the implementation of vaccination, RHDV2 continued to circulate in the premises and to cause sporadic deaths. A nearby, semi-intensive, RHDV2 infected farm belonging to the same owner was identified as the most probable source of the virus. The main risk factors for virus introduction in these two industries were identified. Despite the virus being able to infect a few of the vaccinated rabbits, the significant decrease in mortality rate observed in vaccinated adult rabbits clearly reflects the efficacy of the vaccination. Nonetheless, the time taken to control the infection also highlights the importance of RHDV2 vaccination prior to the first contact with the virus, highly recommendable in endemic areas, to mitigate the infection’s impact on the industry.

Cytoplasmic expression of a thermostable invertase from Thermotoga maritima in Lactococcus lactis.

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Pek, Han Bin; Lim, Pei Yu; Liu, Chengcheng; Lee, Dong-Yup; Bi, Xuezhi; Wong, Fong Tian; Ow, Dave Siak-Wei

2017-05-01

To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis. The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively. Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.

Thermostating extended Lagrangian Born-Oppenheimer molecular dynamics.

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Martínez, Enrique; Cawkwell, Marc J; Voter, Arthur F; Niklasson, Anders M N

2015-04-21

Extended Lagrangian Born-Oppenheimer molecular dynamics is developed and analyzed for applications in canonical (NVT) simulations. Three different approaches are considered: the Nosé and Andersen thermostats and Langevin dynamics. We have tested the temperature distribution under different conditions of self-consistent field (SCF) convergence and time step and compared the results to analytical predictions. We find that the simulations based on the extended Lagrangian Born-Oppenheimer framework provide accurate canonical distributions even under approximate SCF convergence, often requiring only a single diagonalization per time step, whereas regular Born-Oppenheimer formulations exhibit unphysical fluctuations unless a sufficiently high degree of convergence is reached at each time step. The thermostated extended Lagrangian framework thus offers an accurate approach to sample processes in the canonical ensemble at a fraction of the computational cost of regular Born-Oppenheimer molecular dynamics simulations.

Pathogenesis of Dengue Vaccine Viruses in Mosquitoes.

Science.gov (United States)

1980-01-01

1973). Sabin (1948) showed that attenuated dpngiie, passed through mosquitoes, did not revert to pathogenicity frnr man. -7- Thus even if the vaccine ...AD-A138 518 PATHOGENESIS OF DENGUE VACCINE YIRUSES IN MOSQUITOES 1/ (U) YALE UNIV NEW HAVEN CONN SCHOOL OF MEDICINE B J BEATY ET AL. 9i JAN 80 DRND7...34 ’ UNCLASSIFIED 0{) AD 0Pathogenesis of dengue vaccine viruses in mosquitoes -First Annual Report Barry I. Beaty, Ph.D. Thomas H. G

Exploratory field study on the effect of Porcine circovirus 2 (PCV2) sow vaccination on serological, virological and reproductive parameters in a PCV2 subclinically infected sow herd.

Science.gov (United States)

Oliver-Ferrando, Salvador; Segalés, Joaquim; López-Soria, Sergio; Callén, Antonio; Merdy, Olivier; Joisel, François; Sibila, Marina

2018-04-16

This study sought to evaluate the effect of sow vaccination against Porcine circovirus 2 (PCV2) on reproductive parameters during two consecutive reproductive cycles. The study was performed in a PCV2 subclinical infected breeding herd (PCV2 circulation but absence of major reproductive problems). Ninety-four pregnant sows were primo-immunized with a commercial PCV2 vaccine and ninety-seven were injected with phosphate-buffered saline at 6 and 3 weeks before the first studied farrowing, and then boosted at 2 weeks before the second one. Blood samples were taken throughout the study to assess PCV2 DNA load and antibodies. At farrowing, main reproductive parameters and piglet vitality index were registered. In addition, in those litters with more than three mummified or stillborn piglets, microscopic examination and PCV2 antigen detection in foetal myocardium was done. Vaccinated sows showed significantly higher antibody levels compared to the non-vaccinated counterparts. PCV2 DNA was only detected at farrowing in 2 (4.2%) non-vaccinated sows. Vaccinated sows had 1.3 more live-born piglets per litter at the second cycle than non-vaccinated counterparts. Piglets from vaccinated sows had significantly higher (+ 12.7%) vitality score than the ones born from non-vaccinated sows. No PCV2 compatible lesions neither PCV2 antigen were detected in the tested foetal hearts. The present study represents a first attempt to demonstrate that PCV2 sow vaccination may have a positive influence on prolificacy and vitality of the offspring in a subclinical infected breeding herd. However, since reproductive outcomes at farm level may be affected by a number of factors, further studies would be needed to confirm this association.

A Method for Individualizing the Prediction of Immunogenicity of Protein Vaccines and Biologic Therapeutics: Individualized T Cell Epitope Measure (iTEM

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Tobias Cohen

2010-01-01

Full Text Available The promise of pharmacogenomics depends on advancing predictive medicine. To address this need in the area of immunology, we developed the individualized T cell epitope measure (iTEM tool to estimate an individual's T cell response to a protein antigen based on HLA binding predictions. In this study, we validated prospective iTEM predictions using data from in vitro and in vivo studies. We used a mathematical formula that converts DRB1∗ allele binding predictions generated by EpiMatrix, an epitope-mapping tool, into an allele-specific scoring system. We then demonstrated that iTEM can be used to define an HLA binding threshold above which immune response is likely and below which immune response is likely to be absent. iTEM's predictive power was strongest when the immune response is focused, such as in subunit vaccination and administration of protein therapeutics. iTEM may be a useful tool for clinical trial design and preclinical evaluation of vaccines and protein therapeutics.

Reduced antibody responses against Plasmodium falciparum vaccine candidate antigens in the presence of Trichuris trichiura

DEFF Research Database (Denmark)

Esen, Meral; Mordmüller, Benjamin; de Salazar, Pablo Martinez

2012-01-01

BACKGROUND: Helminth infections are highly prevalent in the tropics and may have an effect on immune responses to vaccines due to their immunomodulatory effect. The prevalence of helminth infections in young children, the target group for malaria and most other vaccines, is high. Therefore we...... assessed the influence of helminth infection on vaccine-induced immune responses in a phase I clinical trial of the malaria vaccine candidate GMZ2. METHODS: Twenty Gabonese preschool-age children were vaccinated with GMZ2, a blood stage malaria vaccine candidate. Humoral immune response against the vaccine...... antigens and parasitological status were assessed. Vaccine-specific antibody concentrations and memory B-cell numbers were compared in worm infected and non-infected participants. RESULTS: Antibody response to GMZ2 was 3.4-fold (95% confidence interval: 1.6, 7.4) higher in Trichuris trichiura negative...

Beninese vaccination clinic

OpenAIRE

Linda Sun

2017-01-01

This photo was taken in the village of Ladji, which is on the outskirts of Cotonou, the capital of Benin. At the time, I was a second year medical student volunteering at a local medical clinic. On every Wednesday morning, many Beninese babies, like this one, cry out of discomfort while receiving their monthly vaccinations. The photo shows a local clinic nurse administering the vaccination.

A randomized controlled Phase Ib trial of the malaria vaccine candidate GMZ2 in African children

DEFF Research Database (Denmark)

Bélard, Sabine; Issifou, Saadou; Hounkpatin, Aurore B

2011-01-01

GMZ2 is a fusion protein of Plasmodium falciparum merozoite surface protein 3 (MSP3) and glutamate rich protein (GLURP) that mediates an immune response against the blood stage of the parasite. Two previous phase I clinical trials, one in naïve European adults and one in malaria-exposed Gabonese ...... adults showed that GMZ2 was well tolerated and immunogenic. Here, we present data on safety and immunogenicity of GMZ2 in one to five year old Gabonese children, a target population for future malaria vaccine efficacy trials....

The Vaccination Kuznets Curve: Do vaccination rates rise and fall with income?