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Deborah Voigt and Susan Graham Head All-Star Cast Under Fabio Luisi in the Met's LES TROYENS
Francesca Zambello's new staging of Les Troyens was given its premiere to rave reviews at the Metropolitan Opera in 2003, when the New York Times called it "a visually stunning, seamlessly flowing, and emotionally involving realization of Berlioz's inspired…retelling of Virgil's Aeneid." Now, almost a decade later, the French composer's monumental Trojan War epic returns to the Met for its first revival, opening on December 13 for seven performances.
Led by Met Principal Conductor Fabio Luisi, the revival boasts grand-scale musical forces and a stellar cast headed by soprano Deborah Voigt, reprising her star turn as Trojan prophetess Cassandra, the role in which she triumphed in the production's opening run; mezzo Susan Graham singing her first Met performances as Berlioz's Dido, doomed queen of Carthage; and tenor Marcello Giordani making his house role debut as her lover, Aeneas of Troy. The December 23 and 26 performances will be streamed live on the Met's website, www.metopera.org, and January 5, the closing night, will be transmitted live to movie theaters in the Met's Live in HD series, now seen in almost 2,000 cinemas in 64 countries around the world.
Berlioz himself considered Les Troyens ("The Trojans," 1858) "a great work, greater and nobler than anything done hitherto," but almost a full century would pass before the opera was staged in its entirety. As one of his biographers reports, it was all too often dismissed as "a great sprawling white elephant,…beautiful in patches but…quite unstageable…because of its length." The triumph of Zambello's production was in helping to put this negative assessment to rest. Praising "its lush melodies, nervous rhythms, and heroic marches" the Associated Press judged Les Troyens "an enthralling,…amazing work." In the New York Times, Anthony Tommasini confessed: "All the contrary strands of Berlioz's work come together miraculously in this music. … In this production and this performance, it seemed an inexorable work of genius."
Despite the excellence of the original Met/Zambello cast, Deborah Voigt's performance as the cursed visionary Cassandra was repeatedly singled out for praise. "Voigt used her dramatic soprano with great artistry, unleashing fearsome torrents of sound in her hysterical outbursts, yet reining in her voice during her few tender moments," observed the Associated Press. The Baltimore Sun added: "Voigt's gleaming soprano made Cassandra sympathetic and arresting," while the New York Times concluded: "Ms. Voigt brought her customary blend of burnished power and pliant lyricism to her work. And Zambello has emboldened her to give a physically rigorous and emotionally unfettered performance."
Grammy Award-winner Susan Graham has been described as "unbeatable in French repertoire" (Time Out New York), and according to the Associated Press, she "has the ideal voice for Berlioz." Like Voigt, the mezzo is no stranger to the role she will inhabit in the Met's revival, having already been celebrated for her portrayal of Berlioz's Dido at the Paris Châtelet, where it was recorded live.
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The undead were on the move. Youthful zombies in shredded clothing, with dark-shadowed eyes and a lolling gait shuffled around the audience members, groaning creepily. This 'welcoming party' set the tone for what was to come: a performance of Night Of The Living Dead for Kids, with lashings of strawberry string.
Skeptics might think that the bloody gore and terror of seminal zombie movie NOTLD wouldn't transfer well to a play starring teenagers and performed to their friends and families. And while I wouldn't recommend it to under-10s, as director, soundman and drama teacher Chris Lambert, who adapted the film script for the stage, said: "The students like being able to scare grown-ups." And they're rather good at it, too.
The play opens as Barbara (Erin Tingle) and her brother Johnny (Anthony Thompson) take the long drive into the country to visit their father's grave, only to stumble upon a zombie apocalypse in the making. With Johnny lost to the zombie's clutches, Barbara is pursued to an abandoned house, where she stumbles across the play's hero-figure, Ben (Jack Winstanley), sheltering with the combative Harry (drama teacher Mr Neill), his wife Helen (Amy Rushent), their daughter Karen (Katie Preece) and couple Judy and Tom (Nicole Carslake and Matthew Smith). The tension is ratcheted up with clever use of sound, lights, and intersecting TV and radio news reports (with an entertaining turn from Adrian Rodriguez as the gung-ho zombie-killing expert McClelland), as the plague spreads and the undead slowly surround the house.
Genuinely chilling at points, as zombies shuffled among the audience, explosions shook us and a creepy soundtrack filled the theatre, Night of The Living Dead for Kids remained faithful to the film's pessimistic ending - with a quirky twist you'll just have to see for yourself. And as I drove home in the evening gloom, rain lashing on the windscreen, I glanced in my rearview mirror and tried to put thoughts of shuffling ghouls out of my mind...
The production is at New Greenham Arts Auditorium, Newbury, on Thursday and South Street Arts Centre, Reading, on Friday. The Reading show is sold out.
Book tickets for the New Greenham Arts in Newbury show on Thursday night by calling 01635 522 733.

subset: commoncrawl


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A Melbourne engineering company has agreed to pay $150,000 to the Business Software Alliance (BSA) for allegedly using unlicensed copies of Adobe, Microsoft and Autodesk software.
The case was settled out of court for the amount that included compensatory and punitive damages under Australia's Copyright Act.
Terms of the settlement also called for the engineering firm to purchase or uninstall the unlicensed software and implement software asset management procedures that were approved by the BSA.
BSA Australia co-chair Clayton Noble said the parties disagreed on the exact number of licenses that had been infringed but BSA believed that a "high degree of piracy" had taken place.
The alliance accused the engineering firm of using more than 50 copies of Microsoft software, more than 20 copies of Autodesk's AutoCAD software and five copies of Adobe Acrobat without licenses.
The unnamed engineering company also had two Microsoft Windows servers with a large number of unlicensed users, the BSA claimed.
Noble said the BSA was alerted to the infringements in October by a former company employee, who was frustrated and "unhappy with the way the company refused to get legal".
The informant received $5000 for providing the information through BSA's website. The BSA promised informants up to $US1,000,000, depending on the settlement their information led to.
Copyright © iTnews.com.au . All rights reserved.
Issue: 339 | June 2015
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Q&A with Taryn Manning
For those of you who haven't checked out Jenji Kohan's excellent women's-prison dramedy yet (seriously, what did you do with your summer? go outside?), the holiday TV slump is the perfect time to play catch-up. We talked to Taryn Manning, the actor behind Bible-thumping meth head Tiffany "Pennsatucky" Doggett, about how she gets into character (warning: spoilers ahead).
How do you approach playing a layered villain like Tiffany?
I did a lot of research. I was really never a quote-unquote Method actor—I work more off of instinct. But with [Tiffany], I had to do some next-level research and truly transform. She's so different from me morally. Sometimes when I go home, I can't shed her.
It's definitely a role for a character actor. Is that something you identify yourself as?
In Hollywood terms, I never get to go in as "the attractive girl" anyways. It was tough growing up; I never really understood it. "What do you mean they don't see me like that?" It hurt, you know? At first, I tried to rebel against it. Like, no. I'm turning down any role that's, you know, the drug addict or the hooker or the troubled pregnant girl. And then I did play a couple girl-next-door [parts], and I just didn't have as much fun. So I've embraced it.
Any hints you can give us about season two?
I'm sworn to secrecy. But I can say that I'm back, and I know a lot of people weren't sure whether I lived or died after that last scene [in the season finale]. And you'll see a lot more character development. That's the beauty of the show—you care. You wanna know, why is she here? What's up with the guard?
What's the vibe like on set?
It's been really interesting, [especially] with the costumes. Potato sacks, I call them. [Laughs] It's the great equalizer. With so many women, [the atmosphere could] tend toward cattiness. But we all get along so well, and there's so much support and love. No one gets to do their hair or makeup, so there's no competition. It's a good energy.
Season two of Orange Is the New Black airs on Netflix in 2014.
Source: timeout.com


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Bower Project
the bower project
an interactive, site-specific installation comprised of bamboo, cable ties, microcontrollers, sensors, vibrating motors, brass bells, pools of living moss
This invitational work was created in response to the ordinary and everyday theme of the exhibition, Ordinarily Here, at the Weisman Art Museum in Minneapolis. My installation, the bower project, transformed the everyday experience of walking through a fifty-foot long corridor gallery that led people into the main galleries of the museum. This transitional architectural space was a frequent site of exhibitions, yet it's spatial gesture beckoned people forward, to pass through rather experience the space.
Heightening ones sense of the passage, the bower project re-shaped spatial, aural, and kinesthetic experience. Lengths of bamboo reach up into the sixteen-foot tall entrance area. Vaulting above people as they entered, the bamboo elements reformed the rectilinear volumes so familiar to us in most of our daily encounters with architecture. These bamboo insertions invited people to look anew at this environment and their movement through it.
Positioned as a procession of paired, arching forms, each thicket of bamboo is made of variable sizes and fastened together with vibrant blue cable ties that punctuate the energetic lines of the structures. Inspired by the bowerbird's grass constructions, which they build as courtship lures, this bamboo passageway mimics the allure of the bower. Interactive, kinetic, elements, powered by micro-computers, sensors and vibrating motors, are nested within the thickets of bamboo. These technologies activate the bower's sonic atmosphere. The occasional percussive sound of bamboo rapping upon bamboo called out as people walked through and the sheen of small bells ringing in response to people's movement enlivened the experience of the passage as well.


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USDA chief economist says farm bill will affect income, not plantingsThe U.S. Department of Agriculture increased its estimate of U.S. agricultural exports to $142.6 billion for fiscal 2014, up $5.6 billion from November's estimate and $1.5 billion higher than the previous record level in fiscal year 2013.
By: Jerry Hagstrom, Agweek
The U.S. Department of Agriculture increased its estimate of U.S. agricultural exports to $142.6 billion for fiscal 2014, up $5.6 billion from November's estimate and $1.5 billion higher than the previous record level in fiscal year 2013.
In his annual address Feb. 20 at USDA's annual Agricultural Outlook Forum being held in Arlington, Va., USDA Chief Economist Joseph Glauber noted that the forecast varied by product.
"The forecast for grain and feed exports is boosted $3.2 billion from November to $31.3 billion on greater volumes of wheat, corn and feeds and fodders," he said.
"Oilseed and product exports are forecast at $31.4 billion, up $2.5 billion, driven by record soybean and near-record soybean meal exports. The soybean export forecast is raised $1.8 billion to $21.7 billion as strong demand from China add to both volume and unit value. Higher unit values have increased the cotton export forecast by $100 million to $4.4 billion."
"The export forecast for livestock, poultry and dairy is lowered by $100 million to $31.6 billion, with reductions to poultry, pork and other livestock products outweighing gains to dairy and beef. The horticultural product exports remain unchanged from the record November forecast of $34.5 billion."
Glauber also noted that U.S. agricultural imports for fiscal year 2014 are forecast at $110 billion, up slightly from the November estimate. This forecast is 5.9 percent greater than the fiscal 2013 import total and reflects the relatively weak to moderate recovery of import demand.
"The forecast trade balance in fiscal 2014 is up $5.1 billion from November to $32.6 billion, but still trails the fiscal 2013 surplus of $37.1 billion," he said.
Speaking of U.S. production and farm income, Glauber said the new farm bill will have a "minimal" impact on crop farmers' decisions about what to plant, but the elimination of direct payments has already had an impact on the decline in farm income in 2013.
Glauber said the farm bill will force farmers to make choices between the Agricultural Risk Coverage and Price Loss Coverage programs, but "Since both the PLC and ARC programs are based on producers' base acres rather than on their actual planted acres, the programs will likely have limited impact on acreage decisions."
But Glauber also noted that net cash income for 2014 is forecast at $101.9 billion, down almost 22 percent from 2013, but still more than $5 billion above the previous 10-year average. Most of the decline is a result of the decrease in crop receipts, he said, but the elimination of the $4.9 billion in direct payments that crop farmers have been getting whether prices are high or low also played a role, he told the audience.
Other key points Glauber made:
•The elimination of direct payments under the Agricultural Act of 2014 results in a projected 45 percent decline in government payments. Any expected payments under the new ARC and PLC program would not be paid until calendar 2015. But net indemnities under the crop insurance program have increased in recent years and are expected to exceed $5 billion in 2014.
•Global grain and oilseed consumption has grown faster than the population in the past 10 years and demand for those products is expected to be at record levels in 2014.
Global grain consumption grew by 2.1 percent per year in the past 10 years, compared with a global population growth rate of only 1.2 percent per year. The growth rate varies by grain.
For example, global rice consumption grew at 1.4 percent per year; wheat use grew at 1.8 percent per year and global corn use grew at 3.8 percent per year. Corn use was boosted by industrial use for corn, including ethanol, particularly in the U.S. and China. Oilseed demand growth over the period was even more impressive, growing at more than 3.6 percent annually, with global soybean demand growing at 4.4 percent annually in the past 10 years.
•The biggest wild card in agriculture for 2014 is the drought in the Western states.
•Global trade is expected to increase in the next 10 years by 15 percent for wheat, more than 30 percent for corn and almost 40 percent for soybeans, but U.S. agriculture will face intense competition in export markets from Brazil in soybeans and corn and the Black Sea countries in wheat and corn.
•Despite proposed changes to the Renewable Fuel Standard, corn use for ethanol will remain at 5 billion bushels in 2014 and 2015 because of strong export demand.
•Despite the drought of 2012 and record prices for many commodities, food inflation remained low in 2013. Prices for food consumed at home rose, on average, only 0.9 percent. But meat and poultry prices will likely increase by an estimated 3 to 4 percent in 2014, which will help push food inflation back toward more historical levels.
Tags: farm bill, livestock, crops, agribusiness, updates


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American Idol
American Idol Cute Pic of the Day: Carly Smithson Shows Off Newborn Baby Girl!
Carly Smithson is officially a mommy!
The twenty-nine-year-old American Idol Season 7 alum, who first announced her pregnancy with hubby Todd in June, gave birth to her little one on September, 30 at 10:55 pm.
Carly shared this first-glimpse at daughter Olivia Mabel Smithson via Twitter. Little Olivia apparently took her time coming into the world, as the Irish songstress believed she was due around September 20.
Congrats to Carly and her growing family!
Source: Twitter, Twitter
Need your daily dose of American Idol? Like us on our Facebook page at facebook.com/AmericanIdolFansite, or follow us on Twitter at twitter.com/IdolWetpaint!


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Tim Tebow released by Jets - Rex Ryan basically calls him fat
Tim Tebow is released from the New York Jets -- finally. The team sent the quarterback packing with a rather backhanded compliment from head coach Rex Ryan.
"We have a great deal of respect for Tim Tebow," Ryan says in statement. "Unfortunately, things did not work out the way we all had hoped. Tim is an extremely hard worker, evident by the shape he came back in this offseason (losing weight). We wish him the best moving forward."
There were rumors late in the 2012 season Tebow would return to Florida with the Jacksonville Jaguars as part of a trade deal with the Jets. He now has to pass through waivers, so the Jags could still put in a claim for him if they want to. But, it sounds like they don't. A rep for the team tells USA Today, "The Jacksonville Jaguars' plans do not include Tim Tebow."
The Jets raised the ire of fans throughout the 2012 NFL season by underusing Tebow in favor of failing QB, Mark Sanchez. In just one of the many head-scratching moments from the 6-10 season, Ryan even started third stringer Greg McElroy ahead of Tebow when starter Mark Sanchez was mercifully benched.
But when the former Denver Bronco went to Ryan to plead for his position, haters called him a quitter. "I think that's what's disappointing in the whole situation, people saying, 'Oh, you quit,' or 'You didn't do this,'" Tebow said at the time. "That was not it at all. It was just asking to get an opportunity to play the position I love, which is quarterback."
But he didn't get much of a shot on special teams either, as the plan was said to be. Tebow was left on the bench for the better part of the season. With the Jets taking quarterback Geno Smith in the 2013 draft, Letting go of Tebow brings the team back down to 5 QBs.
If he can't find a spot on an NFL team, Tebow always has options in the Arena Football League. The Orlando predators recently made it known they had a contract waiting for him. Hey, it worked for Kurt Warner.
Photo/Video credit: Getty Images


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holiness in a sentence
Example sentences for holiness
It is a foretaste of what human brain will accomplish when mankind is restored back to holiness.
The saint replied that he was ready to go wheresoever his holiness should think fit.
Whether or not this figure derives its holiness from its symbolic meaning may remain undecided.
What's missing from the performance is a flash of holiness that would make the character a spiritual lightning rod.
Still, there's an intrepid holiness to his prose that redeems his wayward sensibility.
Everything from a robust doctrine of purgatory to dietary laws to the usual holiness tradition stuff.
But they also argue that holiness resides in people, not buildings.
He's able to transform all these essentially simple things into a kind of wholeness and holiness.
On the one side there is the law of gravitation, and on the other the contemplation of the beauty of holiness.
He fretted that it would then be practiced more carelessly-as legal, monogamous marriage is-and its holiness would be diminished.
We scan them for a glimpse of mollifying holiness, and get instead a cruel drumming upon this world's emptiness.
The louder these powers speak about her holiness, the more the general population gets brainwashed into believing a falsehood.
The main point is that someone was able to get her name in the paper and advertise her holiness for all in town to see.
On the one side there is the law of gravitation, and on the other the contemplation of the beauty of holiness.
She has a surgeon's eye for holiness in the quotidian.
Halos are necessary to indicate holiness in pictures and in costumes.
The holiness of these three figures is indicated by the golden halos above their heads.
Famous quotes containing the word holiness
Who is like you, O LORD, among the gods? Who is like you, majestic in holiness, awesome in splendor, doing ... more
I will therefore ... that women adorn themselves in modest apparel, with shamefacedness and sobriety; not with braided h... more
Oh, the holiness of always being the injured party. The historically oppressed can find not only sanctity b... more
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Nearby Words


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Just a day after Hallyu actor Ryoo Si-won's pending divorce was made public, on April 10, an attorney for Ryoo's wife has spoken about the terms that the former wife wants as well as other details.
"For the reason for the divorce, Jo Soo-in decided to leave it blank, as a sign of respect toward her celebrity former spouse. The speculation that she asked for the divorce because he is a drag racer is untrue", said the attorney.
"As a mother, of course she believes that custody of the couple's child should remain with her. Thus she has asked for full custody, this is the condition that she prized above all else".
The lawyer also said that it is possible that the couple may come to an agreement that suits both parties, "depending on how Ryoo reacts".
The former wife of the entertainer is currently residing with her parents and the couple's daughter.
By Carla Sunwoo [email@example.com]
Source : koreajoongangdaily.jo...
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Liverpool target Caner Erkin set to extent contract with Fenerbahce
2014-02-13 06:42:31
Fenerbahçe left-back Caner Erkin will extend his contract at the end of the season according to board member Ömer Temelli.
"He [Caner] met with the president and will renew his contract at the end of the season," Temelli said.
Caner is however, free to speak to other clubs this month, with his contract set to expire at the end of the season.
The Turkish international is being scouted by a number of Premier League clubs including Liverpool according to his agent Batur Altıparmak.
"A few Premier League clubs have been scouting Caner and a few are interested," Batur Altıparmak said speaking to Turkish-football.com.
"However, no offers have been made yet."
Erkin joined CSKA Moscow for €3 million aged 19 and later went onto play for Galatasaray on loan after which he joined Fenerbahçe on a permanent move for €3 million in 2010.
The 25-year-ol has provided the highest number of assists in the Super League (11) this season and also has two goals to his name.


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Slice the rectangle with a hole in its centre into 2 sections so that you could make a square 8x8 (without that hole in the centre).
This old topic is locked since it was answered many times. You can check solution in the Spoiler below.
Pls visit New Puzzles section to see always fresh brain teasers.


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Students will be required to maintain a diary of their weekly activities and submit a comprehensive report of their observations
upon completion. Students will meet at least once during the semester to compare field experiences and submit paperwork.
Prerequisite: None
Corequisite: None
Recommended Preparation: 225 hours paid or 180 hours unpaid work experience Field work in management.
3 UNITS; 3 hours lecture


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Insurance Disputes
If you file a claim against your insurance company and they are not complying with your request, you may have a claim for an insurance dispute. If payment is being denied or your insurance company offers you a subpar payment on a credible claim, contact an experienced insurance dispute attorney.
Types of Insurance Disputes:
– Auto insurance claims
– Medical insurance claims
– Property insurance claims
– Homeowners insurance claims
– Theft claims
– Stolen vehicle claims
Joey Gilbert & Associates aggressive trial attorneys will fight for you to get you the compensation you deserve.
Never a fee unless we win your case!
Contact us today for a free consultation and case evaluation. We are available 24/7 at 775-284-7700 or Toll Free 888-640-4313.


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Dcs Engineering, LLC filed as a Domestic Limited Liability Company (LLC) in the State of Texas on Friday, December 10, 2010 and is approximately five years old, according to public records filed with Texas Secretary of State. The filing is currently active as of the last data refresh which occured on Saturday, July 4, 2015.
Darren Strozewski serves as the Director
Darren Strozewski is also the registered agent for the company. Also known as a statutory or resident agent, the registered agent is responsible for receiving legal notifications regarding court summons, lawsuits, and other legal actions involving the corporate entity.
Key roles for Dcs Engineering, LLC
Darren Strozewski
Director, President
|Filing Type:
|Domestic Limited Liability Company (LLC)
|State ID:
|Date Filed:
|Friday, December 10, 2010
|Registered Agent
|Darren Strozewski
Comprehensive Insight Plus Report
|Learn More
|The D&B Comprehensive Insight Plus Report for Dcs Engineering, LLC combines D&B's proprietary statistical scoring with business, payment and financial information all in one report.
US Company Profile Report
|Learn More
|The D&B U.S. Company Profile contains basic information such as address and telephone number, executive names and titles, line of business and SIC codes, D-U-N-S Numbers, organization status, and year and location of incorporation for Dcs Engineering, LLC.


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The annual Mission Award ceremony recognizes individuals or groups of students, faculty, adjuncts, staff and alumni who have excelled in one of four categories: Living the Mission, Excellence in Professional Preparation, Scholarship Engagement and Societal Reform and Service and Community.
Click here for Mission Award Nomination Form
Past winners are listed here.
Living the Mission Award
The University of St. Thomas School of Law, as a Catholic law school, is dedicated to integrating faith and reason in the search for truth through a focus on morality and social justice. One student from each class and a graduate(s) are selected to receive the "Living the Mission Award." The award is given to the student/graduate who best exemplifies the mission by his or her activities in and/or outside the classroom. The student recipients of this honor also receive cash awards.
Excellence in Professional Preparation
As a law school, we are called to prepare students to be excellent lawyers and professionals – to be servant leaders in their communities. The Mission Award for Excellence in Professional Preparation recognizes those members of the UST Law community who have done an excellent job in preparing our students to become professionals by providing instruction in or demonstrating or fostering ethical principles, character, faith-based perspective, and/or global perspective to enhance our students' knowledge and skill in the practice of law.
Scholarly Engagement and Societal Reform
As an academic institution focused on the law, morality and justice, we are called to engage the broader community on legal issues and policy topics that impact our society. The Mission Award for Scholarly Engagement and Societal Reform recognizes members of the UST Law community who integrate faith, law, ethics, public policy, and social justice into scholarly or advocacy projects which seek improvement in the law, legal institutions, or society.
Service and Community
We are called by our profession and our respective faiths to live our lives "in community," oriented towards serving the disadvantaged, underserved, and those without access to justice. Inspired by Catholic social teaching, the USTLawSchoolMission emphasizes our responsibility to pursue social justice by drawing upon our unique backgrounds and respective beliefs. The Mission Award for Service and Community recognizes members of the UST Law community who have shown exceptional service or leadership through living out our Mission's commitment to improving the lives of members within our law school community and/or in our local, parish, cultural or global communities.


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Profiles: Connecticut's War Dead
A list of military personnel with ties to Connecticut who have died in Iraq and Afghanistan.
April 16, 2012
Spc. Philip C. S. Schiller
Even after he was wounded by enemy fire in Afghanistan, Spec. Philip C. S. Schiller kept firing, his father and his aunt said Monday.
10:23 AM EST, February 10, 2012
Brig. Gen. Terence J. Hildner
Brig. Gen. Terence J. Hildner, a New Haven native who died in Afghanistan on Feb. 3, 2012.
9:19 AM EDT, August 8, 2011
Navy SEAL Brian Bill
Navy SEAL Brian Bill was one of 30 U.S. troops killed on August 6, 2011 when a Chinook helicopter crashed after being fired on in Afghanistan.
10:30 AM EDT, November 1, 2011
Staff Sergeant Ari R. Cullers
Ari R. Cullers, a 28-year-old soldier from Waterford, has died while serving in Afghanistan.
9:50 PM EDT, July 10, 2011
Paul Protzenko
Paul Protzenko, 46, of Enfield, who retired from the state police in 2009, died in Afghanistan after he was struck by small arms fire.
August 15, 2011
Sgt. Edward Jeffrey Frank II
A Hartford native was among five soldiers who died in Afghanistan Thursday when a roadside bomb detonated near their vehicle.
April 28, 2011
Air Force Maj. Raymond G. Estelle II
A 41-year-old New Haven native was one of nine men killed in a shooting at an Afghanistan airport on Wednesday, according to family members
9:48 AM EDT, June 16, 2011
Pvt. Eric D. Soufrine
Woodbridge native Eric D. Soufrine died while on a tour in Afghanistan, according to the Department of Defense. He was 20.
March 31, 2011
Army Staff Sgt. Frank E. Adamski III
Army Staff Sgt. Frank E. Adamski III, 26, of Plainfield, was among three soldiers who died of injuries they suffered when their unit was attacked with small arms. He died March 31, 2011 in Afghanistan.
March 14, 2011
Sgt. 1st Class Dae Han Park
A soldier who graduated from Watertown High School has died in Afghanistan, the Armysaid Monday.
7:25 PM EDT, June 1, 2011
Army Specialist Richard Charlton Emmons III
Richard Charlton Emmons III, 22, an army specialist from Granby, was killed in action in Afghanistan after his vehicle was hit by a rocket propelled grenade.
6:14 PM EST, March 1, 2011
Spc. David R. Fahey Jr.
Spc. David R. Fahey Jr., 23, was killed in action February 28, 2011, in Kandahar Province, Afghanistan.
2:36 AM EDT, September 27, 2010
Pfc. Gebrah Noonan
Gebrah Noonan, an Army private from Watertown, died in Iraq on September 23, 2010.
6:25 PM EDT, August 24, 2010
Sgt. Steven J. DeLuzio
Steven DeLuzio, 25, of Glastonbury, died August 22, 2010, while serving a tour of duty in Afghanistan.
May 27, 2010
Sgt. Edwin Rivera
Sgt. Edwin Rivera died May 27, 2010 at the National Naval Medical Center in Bethesda, Md., of wounds he received in a firefight in Laghman Province on May 20.
April 4, 2010
Lance Cpl. Tyler Owen Griffin
Tyler Owen Griffin, a 19-year-old Marine lance corporal from Voluntown, was recently killed in Afghanistan.
December 31, 2009
Sgt. 1st Class Ronald Jay Spino
As a paratrooper and nurse, Sgt. 1st Class Ronald Jay Spino's job was to help the wounded.
December 11, 2009
Cpl. Xhacob LaTorre
On Monday, in his Texas hospital room, the gravely ill Cpl. Xhacob LaTorre of Waterbury received a Purple Heart for the wounds he suffered in August from a roadside bomb in Afghanistan.
October 5, 2009
Capt. Benjamin A. Sklaver
Capt. Benjamin A. Sklaver of Hamden died Oct. 2, 2009 in an ambush while on patrol in Muscheh, Afghanistan near the Pakistan border. He was 32.
June 29, 2009
Sgt. Edward C. Kramer
Sgt. 1c Edward C. Kramer, 39, of Southington, died in Iraq on June 29, 2009.
September 25, 2008
First Lt. Thomas J. Brown
First Lt. Thomas J. Brown, who grew up in Shelton, was killed in Iraq on Sept. 23, 2008 in Salman Pak, Iraq. He was 26.
1:28 PM EDT, May 29, 2012
Army Specialist First Class Dennis Pratt
Army Specialist First Class Dennis Pratt was killed in an attack in Afghanistan.
June 8, 2008
Cpl. Christian Cotner
Marine Cpl. Christian Cotner of Waterbury died May 30, 2008 in a non-hostile incident in Iraq's Anbar province on his first tour duty in Iraq. Details of his death were not released. He was 20 years old.
October 12, 2007
Sgt. Jason Lantieri
Army Sgt. Jason Lantieri of Killingworth, a member of the Alaska-based 725th Brigade Support Battalion of the 25th Infantry Division, died Oct. 10, 2007 of injuries suffered during vehicle maneuvers. He was 25.
July 8, 2007
Special Warfare Operator Jason D. Lewis
Navy SEAL Jason D. Lewis, a Brookfield native and special warfare operator first class, was killed in combat July 6, 2007 in Baghdad. He was 30 years old, and left a wife and three children in Virginia.
June 28, 2007
Pfc. Andre Craig Jr.
Army Pfc. Andre Craig Jr. of New Haven was killed when a bomb exploded near the convoy on which he was serving as a gunner in Baghdad. He was 24 years old and left an infant daughter.
May 30, 2007
1st Lt. Keith Heidtman
Army 1st Lt. Keith Heidtman was killed May 28, 2007 - Memorial Day - when enemy fire brought down his helicopter in Diyala province northeast of Baghdad. He was 24 years old.
March 25, 2007
Pfc. Orlando Gonzalez
Army Pfc. Orlando Gonzalez, 21, of Bridgeport, died in Iraq on March 25, 2007.
March 23, 2007
Pfc. Stephen K. Richardson
Army Pfc. Stephen K. Richardson, who grew up in Bridgeport, was killed March 20, 2007 by a roadside bomb in Baghdad. He was 22 years old, and left a wife and daughter in Kansas.
February 23, 2007
Sgt. Richard L. Ford
Army Sgt. Richard L. Ford, a decorated soldier from East Hartford, died Feb. 20, 2007 on his third deployment to Iraq of wounds suffered from small arms fire near Baghdad. He was 40 years old.
December 19, 2006
Staff Sgt. Joseph E. Phaneuf II
Army National Guard Staff Sgt. Joseph E. Phaneuf II of Eastford died Dec. 15, 2006 when the armored vehicle he was driving struck a roadside bomb in Afghanistan. He was 38 years old, and left a wife and three children.
November 28, 2006
Capt. Jason Hamill
Army Capt. Jason Hamill, 31, who grew up in Salem, was killed Nov. 26, 2006 with two other soldiers when a roadside bomb exploded in Baghdad. He was within days of finishing his tour in Iraq.
September 6, 2006
Pfc. Nicholas A Madaras
Army Pfc. Nicholas A. Madaras of Wilton, a soldier with the 4th Infantry Division, was killed by a bomb while on foot patrol in Baqouba, Iraq, on Sept. 3, 2006. He was 19 years old.
September 5, 2006
Lance Cpl. Philip A. Johnson
Lance Cpl. Philip A. Johnson of Enfield, a member of the Young Marines from the age of 11, was killed Sept. 3, 2006, by a roadside bomb near Ramadi, Iraq. He was 19 years old.
August 27, 2006
Cpl. Jordan C. Pierson
Marine Cpl. Jordan C. Pierson of Milford, 21, died Aug. 25, 2006 from hostile gunfire while on foot patrol in Fallujah. He was a member of Charlie Company, 1st Battalion, 25th Marines.
May 5, 2006
Cpl. Stephen Bixler
Marine Cpl. Stephen Bixler, a 2003 graduate of Suffield High School, was killed May 4, 2006 while on foot patrol near Fallujah in Iraq. He was 20.
May 6, 2006
Capt. Brian S. Letendre
Marine Capt. Brian S. Letendre, a Virginia native who lived in New Britain for 18 months, was killed in combat in Al Anbar province on May 3, 2006, on his second tour of duty in Iraq. He left a wife and 3-year-old son.
August 4, 2005
Sgt. David Coullard
Marine Sgt. David Coullard, who grew up in Glastonbury, was killed Aug. 1, 2005 by small-arms fire in Haditha, Iraq, with five others in a sniper team. He was 32 years old.
July 1, 2005
Maj. Steve Reich
U.S. Army Major Steve Reich, a West Point graduate and baseball standout from Washington, Conn., died June 28, 2005 with 15 others when militants shot down a helicopter in eastern Afghanistan. He was 34 years old, and had recently married.
June 26, 2005
Army Spec. Christopher Hoskins
Army Spec. Christopher Hoskins of Killingly died June 21, 2005 in Ramadi, Iraq, when his unit came under small arms fire. He had recently re-enlisted. He was 21 years old.
May 16, 2005
Lance Cpl. John T. Schmidt III
Marine Lance Cpl. John T. Schmidt III of Brookfield died May 11, 2005 in an army hospital in San Antonio of combat injuries he suffered Jan. 30, 2005 in Al Anbar province in Iraq. He was 21 years old.
May 11, 2005
Lance Cpl. Lawrence R. Philippon
Marine Lance Cpl. Lawrence R. Philippon of West Hartford was killed by small arms fire May 8, 2005 in Al Anbar province in western Iraq. He was 22 years old.
January 20, 2005
Staff Sgt. Thomas E. Vitagliano
Army Staff Sgt. Thomas E. Vitagliano was killed, along with another member of the 1st Battalion, 503rd Infantry Regiment, by a car bomb on Jan. 17, 2004 in Ramadi, Iraq. He was 33 and had been engaged to marry.
December 15, 2004
Spc. Robert Hoyt
Army Spc. Robert Hoyt, a member of the C Company, 102nd Infantry Battalion from Bristol, was killed Dec. 11, 2004 when his armored vehicle struck a bomb in Baghdad. He was a graduate of E.O Smith High School in Storrs and has family in the Ashford area. He was 21 years old.
December 7, 2004
Staff Sgt. Henry E. Irizarry
Army Staff Sgt. Henry E. Irizarry of Waterbury died Dec. 3, 1004 in Taji, Iraq, when a roadside bomb detonated near his vehicle. He was 38 years old.
December 2, 2004
Lt. Col. Michael J. McMahon
Army Lt. Col. Michael J. McMahon, who grew up in West Hartford and graduated from Conard High School, died Nov. 27, 2004 in a plane crash in the mountains near Bamiyan, Afghanistan. McMahon, 41, left a wife, also a lieutenant colonel, and three children.
November 25, 2004
Sgt. Joseph Michael Nolan
Army Sgt. Joseph Michael Nolan, a Waterbury native and graduate of Wolcott High School, died Nov. 18 in Fallujah. He was assigned to the 312th Military Intelligence Battalion, 1st Cavalry Division, from Fort Hood, Texas. He was 27 years old.
4:02 PM EST, November 25, 2004
Sgt. Joseph Michael Nolan
A military intelligence expert born in Waterbury was killed Nov. 18 in Fallujah when his vehicle was struck by an explosive device, military officials announced Wednesday.
November 16, 2004
Cpl. Kevin J. Dempsey
Marine Cpl. Kevin J. Dempsey of Monroe died Nov. 13, 2004 in an explosion in Al Anbar Province in Iraq. Dempsey, a graduate of New Canaan High School, was in the 2nd Reconnaissance Battalion, attached to the II Marine Expeditionary Force. He was 23.
October 19, 2004
Chief Warrant Officer William Brennan
Army Chief Warrant Officer William Brennan died Oct. 16, 2004 when the helicopter he was flying collided with another over Baghdad. He grew up in Behlehem, Conn., and leaves a wife and two daughters in Hawaii. He was 36 years old.
August 28, 2004
Spec. Jacob D. Martir
Army Spec. Jacob D. Martir, a native of Puerto Rico who grew up in Willimantic and Norwich, was killed Aug. 18, 2004 by small-arms fire while on patrol in a part of Baghdad known as Sadr City. He was 21 years old.
June 9, 2004
Pfc. Melissa Hobart
Army Private First Class Melissa Hobart, who grew up in East Haven, collapsed and died June 6, 2004 while serving guard duty in Baghdad. The cause of her death had yet to be determined. She was 22, and leaves a 3-year-old daughter.
April 9, 2004
Petty Officer Nathan B. Bruckenthal
Coast Guard Petty Officer 3rd Class Nathan B. Bruckenthal, who lived in Ridgefield during his teens, died April 24, 2004 in an attack near the Khawr Al Amaya oil terminal. His was the first combat death for the Coast Guard since the Vietnam War. He was 24 years old.
April 9, 2004
Sgt. Felix Delgreco
Army Sgt. Felix Delgreco of Simsbury, a member of C Company, 102nd Infantry in Bristol, was killed April 9, 2004 while on patrol in Baghdad, when his vehicle struck an improvised bomb and was attacked with small arms. He was the first member of the Connecticut Army National Guard to die in Iraq. He was 22 years old.
April 10, 2004
Spec. Tyanna Avery-Felder
Army Spec. Tyanna Avery-Felder died April 6, 2004, about two days after she was wounded by a roadside bomb in Iraq. She was a cook with the Army's Stryker Brigade, based in Fort Lewis, Wash. She was raised in Bridgeport, and was the first woman from Connecticut to die in Iraq. She was 22 years old.
February 15, 2004
Sgt. Benjamin Gilman
Army Sgt. Benjamin Gilman of Meriden, a combat engineer with the Army's 41st Engineering Battalion, died Jan. 29, 2004, in an accidental explosion while his unit tried to dispose of a weapons cache in Ghanzi, Afghanistan. He was 28 years old.
January 7, 2004
Capt. Eric Paliwoda
Army Capt. Eric Thomas Paliwoda, who grew up in Farmington and West Hartford, died Jan. 2, 2004 in a mortar attack on his base at Balad, Iraq. He commanded Company B of the 4th Engineer Battalion, 3rd Brigade Combat Team, 4th Infantry Division. He was 28 years old.
December 20, 2003
Pfc. Jeffrey Braun
Army Pfc. Jeffrey Braun of Stafford, a member of the 82nd Airborne Division, died December 11, 2003 in Baghdad. The Department of Defense said he died from a "non-hostile gunshot wound." He was 19 years old.
November 26, 2003
Sgt. Maj. Philip Albert
Army Sgt. Maj. Philip Albert, who grew up in Plymouth, died Nov. 23, 2003 a helicopter crash near Bagram Air Base in Afghanistan. He was stationed at Fort Drum in upstate New York with the 10th Mountain Division. He was 41 years old.
November 9, 2003
Pfc. Anthony D'Agostino
Army Pfc. Anthony D'Agostino of Waterbury was one of 16 U.S. soldiers who died after a CH-47 Chinook helicopter was shot down over Iraq Nov. 2, 2003. He was a communications specialist attached to the 16th Signal Brigade in Fort Hood, Texas. He was 20 years old.
August 24, 2003
Sgt. David Travis Friedrich
Army Sgt. David Travis Friedrich of Naugatuck, a member of the 325th Military Intelligence Company in Waterbury, died Sept. 20, 2003 when mortars hit a U.S. base near the Abu Ghraib prison on the western outskirts of Baghdad. He was 26 years old.
August 24, 2003
Staff Sgt. Richard S. Eaton Jr.
Army Staff Sgt. Richard S. Eaton Jr., a counterintelligence analyst who grew up in Guilford, died in his sleep in Ar Ramadi, Iraq, on Aug. 20, 2003. The Army reported that he probably died of pulmonary edema. He was 37 years old.
July 31, 2003
Pfc. Wilfredo Perez Jr.
Army Pfc. Wilfredo Perez Jr. of Norwalk was guarding a children's hospital in Baquouba, 45 miles northeast of Baghdad, when he and two other members of the 4th Infantry Division were killed in a grenade attack on July 26, 2003. He was 24 years old.
April 28, 2003
Cpl. Kemaphoom Chanawongse
Marine Cpl. Kemaphoon ``Ahn'' Chanawongse of Waterford was running to retrieve ammunition when he was killed by an enemy round on March 23, 2003 in a battle in Nasiriyah, Iraq. He was 22 years old.
March 26, 2003
Gunnery Sgt. Phillip Jordan
Marine Gunnery Sgt. Phillip Jordan of Enfield, a Texas native based at Camp Lejeune in North Carolina, was assigned to the 1st Battalion, 2nd Marine Regiment, 2nd Marine Expeditionary Brigade. He died March 23, 2003 in a battle in Nasiriyah, Iraq. He was 42 years old.
March 7, 2002
Tech. Sgt. John A. Chapman
Tech. Sgt. John A. Chapman died on March 4, 2002 along with six other servicemen during a reconnaissance mission in Afghanistan. Chapman, 36, was the first Connecticut native to die in combat in the war in Afghanistan.
Copyright © 2015, The Hartford Courant


text:
The present invention relates to recombinant vaccinia viruses which are derived from the modified virus Ankara (MVA) and which contain a DNA sequence which codes for a foreign antigen. The invention also relates to vaccines which contain a virus of this type in a form suitable for injection, as well as to a method for the preparation thereof. Furthermore, the invention relates to the use of a recombinant vaccinia virus MVA for the preparation of heterologous proteins in eukariotic cells.
1. A modified vaccinia virus Ankara useful in vaccine formulations, said virus comprising a DNA sequence which codes for a foreign antigen without impairing the viability of the MVA, which foreign antigen causes infection or disease.
2. The virus of claim 1 wherein said foreign antigen is malaria parasite Plasmodium falciparum.
3. A vaccine formulation comprising an effective amount of a modified vaccinia virus Anhara comprising a DNA sequence which codes for a foreign antigen without impairing the viability of the MVA, which foreign antigen causes infection or disease, and a physiologically acceptable carrier.
4. A method for the prophylatic protection of humans or animals against an infection caused by a pathogenic agent exhibiting pathogenic properties, which method comprises the use of the vaccine formulation of claim 3.
5. The vaccine formulation of claim 3 wherein said pathogenic agent is malaria parasite Plasmodium falciparum.
Vaccinia viruses belong to the family of poxviruses. Certain strains of vaccinia viruses have been used for many years as live vaccine to immunize against smallpox, for example the Elstree strain of the Lister Institute in the UK. Because of the complications which may derive from the vaccination (compare Schar, Zeitschr. fur Praventivmedizin 18, 41-44 [1973]), and since the declaration in 1980 by the WHO that smallpox had been eradicated nowadays only people at high risk are vaccinated against smallpox.
Vaccinia viruses have also been used recently as vectors for foreign antigens (Smith et al., Biotechnology and Genetic Engineering Reviews 2. 383-407 [1984]). This entails DNA sequences (genes) which code for foreign antigens being introduced, with the aid of DNA recombination techniques, into the genome of the vaccinia viruses. If the gene is integrated at a site in the viral DNA which is non-essential for the life cycle of the virus, it is possible for the newly produced recombinant vaccinia virus to be infectious, that is to say able to infect foreign cells and thus to express the integrated DNA sequence (compare European Patent Applications with the publication Nos. 83,286 [published on Jul. 6, 1983] and 110,385 [published on Jun. 13, 1984]). The recombinant vaccinia viruses prepared in this way can be used, on the one hand, as live vaccines for the prophylaxis of infections which are caused by these antigens or, on the other hand, for the preparation of heterologous proteins in eukaryotic cells. Most of the recombinant vaccinia viruses described in the literature are based on the WR strain. On the other hand, it is known that this strain has a high neurovirulence and is thus poorly suited for use in humans and animals (Morita et al., Vaccine 5, 65-70 [1987]).
However, strains of viruses specially cultured to avoid undesired side effects have been known for a long time. Thus, it has been possible, by serial passages of the Ankara strain of vaccinia virus (CVA) on chicken fibroblasts, to culture a modified vaccinia virus Ankara (MVA) (Swiss Patent No. 568,392). This modified vaccinia virus Ankara has only low virulence, that is to say it is followed by no side effects when used for vaccination. Hence it is particularly suitable for the initial vaccination of immunocompromised subjects. The excellent properties of the MVA strain have been demonstrated in a large number of clinical trials (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167. 375-390 [1978], Stickl et al., Dtsch. med. Wschr. 99, 2386-2392 [1974]).
Hence, a possible way to introduce foreign genes by DNA recombination into the MVA strain of vaccinia virus has been sought. Since the intention was not to alter the genome of the MVA except at the recombination site, it was necessary to use a method which complied with this requirement. Since the thymidine kinase gene (TK gene) of MVA is not of fundamental importance for the life cycle of the virus, the foreign DNA sequence was recombined in the DNA of the vaccinia TK gene.
The present invention relates to recombinant MVA vaccinia viruses which contain a gene which codes for a foreign antigen, preferably of a pathogenic agent, and to vaccines which contain a virus of this type in a physiologically acceptable form. The invention also relates to methods for the preparation of such recombinant MVA vaccinia viruses or vaccines, and to the use of these vaccines for the prophylaxis of infections caused by such antigens or pathogenic agents.
The recombinant MVA vaccinia viruses can be used to prepare heterologous polypeptides in eukaryotic cells. This entails cells being infected with the recombinant vaccinia viruses. The gene which codes for the foreign antigen is expressed in the cells. The expressed heterologous polypeptide can be isolated. The methods to be used for the production of such heterologous polypeptides are generally known to those skilled in the art (compare, for example, European Patent Applications with the publication numbers 206,920 [published on Dec. 30, 1986] and 205,939 [published on Dec. 30, 1986]. The polypeptides produced with the aid of the recombinant MVA viruses are, by reason of the special properties of the MVA viruses, more suitable for use as medicaments in humans and animals.
The recombinant MVA vaccinia viruses can be prepared as set out hereinafter. Eukaryotic cells, preferably mammalian cells, especially CVl monkey cells (obtainable from Flow Laboratories, Catalogue No. 02-240, from the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md., U.S.A., under Depositary No. CCL 70, and from the European Collection of Animal Cell Cultures (ECACC), PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Great Britain, under Depositary No. 85011434; described by Jensen et al., Proc. Natl. Acad. Sci. USA 52, 53-59 [1964]) can be infected with the MVA. MVA and its properties are described in detail in the literature (Mayr et al., supra). The MVA virus was deposited in compliance with the requirements of the Budapest Treaty at CNCM (Institut Pasteur, Collection Nationale de Cultures de Microorganisms, 25, rue du Docteur Roux, 75724 Paris Cedex 15) on Dec. 15, 1987 under Depositary No. I-721. The MVA virus can be grown on chicken embryo fibroblasts (CEF) as described in the example. MVA is distinguished by its great attenuation, that is to say by diminished virulence or infectiosity while maintaining good immunogenicity. The extent of diminution of the virulence can be determined experimentally in a variety of ways, for example by the behaviour of the virus on cell cultures, by measurement of the neurovirulence; or of the virulence on subcutaneous administration, or by a virulence assay in immunosuppressed mice. The highly attenuated MVA strain shows a great change in the behaviour on cell cultures and a neurovirulence which is at least a factor of 107 less than that of the wild-type WR of vaccinia virus. This difference is also evident on comparison of recombinant wild-type viruses of the WR strain with recombinant viruses of the MVA strain.
According to the present invention, a DNA which contains a partial sequence from a non-essential segment of the vaccinia virus, DNA, and a DNA sequence which codes for a foreign antigen, is introduced into the infected cell.
The DNA can be introduced into the cells by transfection, for example by means of calcium phosphate precipitation (Graham et al., Virol. 52, 456-467 [1973]; Wigler et al., Cell 777-785 [1979], by means of electroporation (Neumann et al., EMBO J. 1, 841-845 [1982]), by microinjection (Graessmann et al., Meth. Enzymology 101, 482-492 [1983]), by means of liposomes (Straubinger et al., Methods in Enzymology 101, 512-527 [1983]), by means of spheroplasts (Schaffner, Proc. Natl. Acad. Sci. USA 77, 2163-2167 [1980]) or by other methods known to those skilled in the art. Transfection by means of calcium phosphate precipitation is preferably used.
The inserted DNA can be linear or circular. A circular DNA is preferably used. It is particularly preferable to use a plasmid. The DNA contains at least one partial sequence from a non-essential segment of the vaccinia virus DNA. Non-essential segments of the vaccinia virus DNA are known to those skilled in the art. One example of a non-essential segment of this type is the thymidine kinas gene and its adjacent regions (Weir et al., J. Virol., 530-537 [1983]). The preferred partial sequence is contained in the plasmid pHGS-2/5.1, whose construction is described in the European Patent Application with the publication No. 198,328, published on Oct. 22, 1986.
The plasmid pHGS-2/5.1 contains a gene which codes for the 5.1 antigen of the malaria pathogen Plasmodium falciparum (Hope et al., Nucleic Acids Res. 13, 369-379 [1985]). The DNA can contain, in place of this gene which codes for a malaria antigen, other known genes from pathogenic agents. Pathogenic agents are to be understood to be viruses, bacteria and parasites which may cause a disease, as well as tumor cells which multiply unrestrictedly in an organism and may thus lead to pathological growths. Examples of such pathogenic agents are described in Davis, B.D. et al., (Microbiology, 3rd ed., Harper International Edition). Preferred genes of pathogenic agents are those of the malaria parasite Plasmodium falciparum, of the tuberculosis-causing Mycobacteria, of herpes viruses and of human immunodeficiency viruses, for exampIe HIV I and HIV II.
In order for it to be possible for the DNA sequence or the gene which codes for a foreign antigen to be expressed, it is necessary for regulatory sequences, which are required for the transcription of the gene, to be present on the DNA. Such regulatory sequences (called promoters) are known to those skilled in the art, for example those of the vaccinia 11 kDa gene as are described in European Patent Application, Publication No. 198,328, and those of the 7.5 kDa gene (European Patent Application, Publication No. 110,385).
Once the gene coding for a foreign antigen has been introduced into the eukaryotic cell and the foreign DNA has recombined with the viral DNA, it is possible to isolate the desired recombinant vaccinia virus in a manner known per se, preferably with the aid of a marker (compare Nakano et al., Proc. Natl. Acad. Sci. USA 79, 1593-1596 [1982], Franke et al., Mol. Cell. Biol. 1918-1924 [1985], Chakrabarti et al., Mol. Cell. Biol. 3403-3409 [1985], Fathi et al., Virology 97-105 [1986]). This is preferably carried out, as described in the examples, by adding the infected eukaryotic cells, two days after transfection, to 143B TK- cells (ATCC No. CRL 8303) and incubating the latter in the presence of bromodeoxyuridine (BUdR) for a further two days. During this, all the cells which contain a functional thymidine kinase gene (TK gene) are killed, and only the TK- cells, as well as TK- cells which contain a vaccinia virus genome in which the TK gene has been inactivated by integration of a foreign gene, are now able to survive.
This selection step is preferably carried out several times. In order to obtain single viral clones, it is possible after the BUdR selection to infect primary chicken embryo fibroblasts (CEF) with the cell suspension of 143B TK- cells and to incubate them under an agarose medium as described in the example. The expression of the foreign antigen can be detected in the cloned viruses by means of fluorescent antibodies. Positive clones can be further purified by plaque purification. The recombinant MVA vaccinia viruses obtained in this way are able to express the gene which codes for a foreign antigen and is contained in the viral genome. It has emerged that the expression of such genes is approximately equally strong in recombinants of the greatly attenuated MVA strain and in recombinants of the WR strain customarily used.
To prepare vaccines, the MVA vaccinia viruses according to the invention are converted into a physiologically acceptable form. This can be done based on the many years of experience in the preparation of vaccines used for vaccination against smallpox (Kaplan, Br. Med. Bull. 25, 131-135 [1969]). Typically, about 106 -107 particles of the recombinant MVA are freeze-dried in 100 μl of phosphate-buffered saline (PBS) in the presence of 2% peptone and 1% human albumin in an ampoule, preferably a glass ampoule. The lyophilisate can contain extenders (such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone) or other aids (such as antioxidants, stabilizers, etc.) suitable for parenteral administration. The glass ampoule is then sealed and can be stored, preferably at temperatures below -20� C., for several months.
For vaccination the lyophilisate can be dissolved in 0.1 to 0.2 ml of aqueous solution, preferably physiological saline, and administered parenterally, for example by intradermal inoculation. The vaccine according to the invention is preferably injected intracutaneously. Slight swelling and redness, sometimes also itching, may be found at the injection site (Stickl et al., supra). The mode of administration, the dose and the number of administrations o can be optimized by those skilled in the art in a known manner. It is expedient where appropriate to administer the vaccine several times over a lengthy period in order to obtain a high titre of antibodies against the foreign antigen.
The detailed example which follows is intended to contribute to a better understanding of the present invention. However, it is not intended to give the impression that the invention is confined to the subject-matter of the example.
The MVA virus is a greatly attenuated vaccinia virus produced by serial passages of the original CVA strain on chicken embryo fibroblast (CEF) cultures. For a general review of the history of the production, the properties and the use of the MVA strain of vaccinia, reference may be made to the summary published by Mayr et al. in Infection 3, 6-14 [1975]. Owing to the adaptation to CEF, growth of the MVA virus on other cell systems is greatly restricted, and plaque formation by the virus is now detectable only on CEF cells. Although no plaque formation can be found on monkey CVl (ATCC No. CCL 70) and human 143B TK- cells (ATCC No. CRL 8303; Bacchetti et al., Proc. Natl. Acad. Sci. USA 74, 1590-1594 [1977]), it was nevertheless possible to show, as described hereinafter, that replication of the MVA virus takes place at least on these two cell lines and is approximately comparable to that on CEF cells. The ability of the MVA virus to replicate on CVl and 143B TK- cells was utilized for the recombination of foreign antigens into the viral genome (see below). However, in order not to alter the properties of the MVA virus it was normally grown on CEF cells, the host cell for which it had been adapted. To prepare the CEF cells, 11-day old embryos were isolated from incubated chicken eggs, the extremities were removed, and the embryos were cut into small pieces and slowly dissociated in a solution composed of 0.25% trypsin at room temperature for 2 hours. The resulting cell suspension was diluted with one volume of medium I (MEM Eagle, for example obtainable from Gibco, Basle, Switzerland; Order No. 072-1500) containing 5% fetal calf serum (FCS), penicillin (100 units/ml), streptomycin(100 μg/ml)and 2 mM glutamine and filtered through a cell screen (for example obtainable from Technomara AG, Zurich, Switzerland, Order No. Bellco 1985, 150 mesh), and the cells were sedimented by centrifugation at 2000 rpm in a bench centrifuge (Hermle KG, D-7209 Gosheim, FRG) at room temperature for 5 minutes. The cell sediment was taken up in 1/4 of the original volume of medium I, and the CEF cells obtained in this way were spread on cell culture dishes. They were left to grow in medium I in a CO2 incubator at 37� C. for 1-2 days, depending on the desired cell density, and were used for infection either directly of after 1-2 further cell passages. A clear description of the preparation of primary cultures can be found in the book by R. I. Freshney, "Culture of animal cells", Alan R. Liss Verlag, New York [1983], Chapter 11, page 99 et seq.
MVA viruses were used for infection as follows. CEF cells were cultured in 175 cm2 cell culture bottles. At 80-90% confluence, the medium was removed and the cells were incubated for one hour with an MVA virus suspension (0.01 infectious particles (=pfu) per cell, 0.01 ml/cm2) in phosphate-buffered saline (PBS/Dulbecco, for example Animed AG, Muttenz, Switzerland, Order No. 23.100.10). Then medium I was added (0.2 ml/cm2) and the bottles were incubated at 37� C. for 2-3 days until about 80% of the CEF cells had lysed. The virus lysates were stored with the cells and medium, without treatment, in the cell culture bottles at -30� C. before further processing (purification etc.).
In contrast to the MVA virus, the WR virus (ATCC No. VR-119) is able to grow in virtually all cell lines (Drillien et al., J. Virology 843-850 [1978]) and its growth can be observed directly by means of plaque formation. Thus, although there was no pressing reason to grow this virus on CEF cells, nevertheless these cells were usually chosen in the experiments described hereinafter, in order to eliminate as far as possible factors specific to the host cells in the direct comparison of the two viruses, MVA and WR. Growing was carried out as described for the MVA virus.
The purification steps undertaken to obtain a virus preparation which was as pure as possible and free from components specific to the host cell were identical for the MVA and WR viruses (Joklik, Virology 18, 9-18 [1962], Zwartouw et al., J. gen. Microbiol 29, 523-529 [1962]). The cell cultures which had been infected and then stored at -30� C. were thawed, the residual cells were shaken off or scraped off the plastic substrate, and cells and virus were removed from the medium by centrifugation (Sorvall centrifuge, GSA rotor, 1 hour at 5000 rpm and 10� C.). The sediment, composed of viral and cell particles, was suspended once in PBS (10-20 times the volume of the sediment), and the suspension was centrifuged as above. The new sediment was suspended in 10 times the volume of RSB buffer (10 mM Tris-HCl pH 8.0, 10 mM KCl, 1 mM MgCl2), and the suspension was briefly treated with ultrasound (Labsonic 1510 equipped with a 4 mm diameter tip, obtainable from Bender and Hobein, Zurich, Switzerland; 2�10 seconds at 60 watts and room temperature) in order to disintegrate remaining still intact cells and to liberate the virus particles from the cell membranes. The cell nuclei and the larger cell debris were removed in the subsequent brief centrifugation of the suspension (Sorvall GSA rotor obtainable from DuPont Co., D-6353 Bad Nauheim, FRG; 3 minutes at 3000 rpm and 10� C.). The sediment was once again suspended in RSB buffer, treated with ultrasound and centrifuged, as described above. The collected supernatants containing the free virus particles were combined and layered over a pad composed of 10 ml of 35% sucrose in 10 mM Tris-HCl, pH 8.0, and centrifuged in a Kontron TST 28.38/17 rotor (Kontron Instrumente, Zurich, Switzerland; corresponds to a Beckman SW 27 rotor) for 90 minutes with 14,000 rpm at 10� C.). The supernatant was decanted, and the sediment containing the virus particles was taken up in 10 ml of 10 mM Tris-HCl, pH 8.0, homogenized by brief treatment with ultrasound (2�10 seconds at room temperature, apparatus as described above), and applied to a stepped gradient for further purification. The steps of the gradient were each composed of 5 ml of sucrose in 10 mM Tris-HCl, pH 8.0 (sucrose concentration steps: 20%, 25%, 30%, 35% and 40%). The gradient was centrifuged in a Kontron TST 28.38/17 rotor at 14,000 rpm 10� C. for 35 minutes. After this centrifugation, several discrete zones containing virus particles were visible in the region of the gradient between 30% and 40% sucrose. This region was siphoned off from the gradient (10 ml), the sucrose solution was diluted with PBS (20 ml) and the virus particles were sedimented therefrom by centrifugation (Kontron TST 28.38/17 rotor, 90 minutes at 14,000 rpm, 10� C.). The sediment, which now consisted mostly of pure virus particles (comparison between OD measurement and plaque test, see below), was taken up in PBS in such a way that the virus concentrations corresponded on average to 1-5�109 pfu/ml. The purified virus stock solution was used either directly or diluted with PBS for the subsequent experiments.
The determination of the concentration of the virus preparations was usually carried out in two ways and thus also permitted a statement to be made about the purity of the virus stock. The absolute concentration of virus particles in the solution was determined by measurement of the optical density of the virus solution at 260 nm in a spectrophotometer where 1 OD260 corresponds to a virus concentration of 1.2�1010.spsp.260 particles per ml (Joklik, supra). For this purpose, the above virus solutions were diluted in the ratio 1:100 with PBS, and the absorption was measured in quartz cuvettes with PBS solution as reference. Assuming that only about every sixtieth virus particle from a preparation of this type is able to infect cell culture cells, that is to say is infectious (Joklik, Bacteriol. Rev. 30, 33-66 [1966]), it is possible to compare directly the virus titre calculated from the measurement of optical density and the virus titre obtained by the method described hereinafter.
For titrating out the virus solutions on cell cultures, CEF cultures were grown in medium I in 8 cm2 plastic cell culture dishes. The medium was aspirated from dishes which were 80-90% confluent, and 0.2 ml of virus diluted with PBS was added to each. The dishes were then left to stand at room temperature for 1 hour. The virus solution was diluted in decimal steps. After this incubation at room temperature, 2 ml of agarose medium I (consisting of medium I+1% agarose) were added to each dish, and the dishes were incubated in a CO2 incubator for 16-24 hours. Then a layer Of 2 ml of agarose medium I, which contained in addition 0.2% neutral red for staining the live cells, was Placed on top, and the dishes were incubated for a further 16-24 hours. After this time had elapsed, it was possible to count the colourless plaques under a binocular, and the resulting number of plaques was compared with the value calculated from the optical density. Good agreement was found in most cases. The virus titre calculated from the optical measurement was usually a factor of about 2 to 4 higher.
TABLE I__________________________________________________________________________1/500 1/50 1/5 1:20 1:100 1:500__________________________________________________________________________LM TK- ++ ++ +++ 5/9 0/0 0/0RK13 + + ++++ 10/13 3/3 1/0CV1 ++ +++ ++++++ nd 500/nd 110/140RITA ++ +++++ ++++++ 31/38 10/5 3/0HeLa ++ ++ ++++++ 31/ 39 10/9 1/0AG1523 +++++ +++++ ++++++ 11/8 1/0 0/0143B TK- ++ +++++ ++++++ 156/nd 60/86 30/29 ##STR1##__________________________________________________________________________ (nd = not determined)
The cell lines LM TK- (ATCC No. CCL 1.3), RK13 (ATCC No. CCL 37), CVl (ATCC No. CCL 70), Rita (RC-37, Schroder et al., J. gen. Virol. 41, 493-501 [1978]), HeLa (ATCC No. CCL 2), AG1523 (Human Genetic Mutant Cell Repository, National Institute of General Medical Science NIGMS, Camden, N.J., USA) and 143B TK- (ATCC No. CRL 8303) listed in Table I were spread out in 8 cm2 dishes (12 dishes for each line) and left to grow under medium I to 80-90% confluence in a CO2 incubator at 37� C. The medium was then aspirated off, and the cells were each infected wit 0.2 ml of MVA virus (in PBS), specifically in concentrations of one infectious virus particle (pfu) per 500, 50 or 5 cells, respectively. After 1 hour at room temperature, 2 ml of medium I were added to the cells and the latter were incubated at 37� C. After 2 days, medium was removed from one half of the culture dishes, and the cells were fixed and stained with a solution of 2% neutral red in 18% ethanol (1 ml/dish). No plaques were detectable in any of the stained cultures, but the cells in each culture showed cytopathic effects (cell changes) which could be pronounced (++++) or weak (+) depending on the cell line. High virus concentrations (1 pfu/5 cells) resulted in general cell death (++++++) in most cases. See left-hand side of Table I.
It is not possible to establish from the experiment described above whether growth of the MVA virus has taken Place in the various cell lines or not. For this reason, the medium and the cells from each of the remaining, unstained culture dishes were collected, briefly homogenized with ultrasound, and in homogenates obtained in this way were used to infect CEF cells. The homogenates were diluted with PBS in the ratio 1:20, 1:100 and 1:500. 0.2 ml of each of the dilutions was added to 2 dishes of CEF cells which were 80-90% confluent and from which the medium had previously been removed. The cells were incubated at room temperature for one hour and then a layer of agarose medium I was placed on top (2 ml/dish). After incubation at 37� C. for 16-24 hours, agarose medium I containing 0.2% neutral red was added (2 ml/dish) and the dishes were incubated for a further 16-24 hours. The colourless plaques were now readily visible and were counted (pfu/dish). The results for the individual cell lines are recorded in the right hand side of Table I. These are the figures for the dilutions of the intermediate virus concentrations (1 pfu/50 cells). From the original number of MVA virus particles used (pfu/cell), the dilution of the homogenates and the volume used for infection of the CEF cells it is possible to calculate the number of virus particles which were placed on the CEF (=amount of virus added), assuming that the MVA virus had not grown in the individual cell lines. It is evident from the table that the number of plaques was significantly above the figure expected from the amount of virus added only in two of the tested cell lines (CVl and 143B TK-), that is to say measurable growth of the MVA virus had taken place only in these two cell lines.
The foreign antigen -was inserted into the vaccinia virus genome by the method described by Mackett et al., (Proc. Natl. Acad. Sci. USA 79, 7415-7419 [1982]) which is based on homologous recombinations in the viral TK gene. The construction of the plasmid pHGS-2/5.1 which was used for the recombination in the present example is described in detail in European Patent Application, Publication No. 198,328, published on Oct. 22, 1986. The starting plasmid pHGS-2 was deposited, in the form of a culture of E. coli HB101 transformed with the plasmid pHGS-2, in compliance with the Budapest Treaty, on Feb. 21, 1985 under Depositary No. DSM 3249 at the Deutsche Sammlung von Mikrooranismen DSM in Gottingen, Germany, and was made freely available on Oct. 4, 1986. The DSM is located in D-3300 Braunschweig at Mascheroder Weg IB since November 1987.
The recombination of the 5.1 antigen in the WR virus was carried out by a protocol which permits double selection for recombinant viruses. In a first step, medium was removed from 8 cm2 culture dishes containing CVl monkey cells which were 80-90% confluent, and then a layer of 0.2 ml of a virus suspension of the temperature-sensitive vaccinia mutant ts N7 (Drillien et al., Virology 131, 385-393 [1983]) was placed on top (0.1 pfu/cell). The dishes were left to stand at room temperature for 1 hour. Then medium I was added (2 ml/dish) to the infected cells, and the dishes were incubated in a CO2 incubator at the temperature of 33� C. which is permissive for the virus, for 2 hours (Kieny et al., Nature 312, 163-166 [1984]). Half an hour before this incubation time had elapsed, a calcium phosphate precipitate containing the DNAs was prepared (Graham et al., supra). For this purpose, 0.55 ml of HeBS buffer (1.5 mM disodium hydrogenphosphate, 280 mM NaCl, 50 mM HEPES adjusted to pH 7.1) was introduced, followed by 200 ng of purified vaccinia WR virus DNA and 200 ng of the purified plasmid pHGS-2/5.1 DNA, each in 1 μl of TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA). 0.55 ml of a 250 mM calcium chloride solution was added dropwise, while agitating gently, and the mixture was left to stand at room temperature for 20 minutes. The medium was then drawn off from the culture dishes, and the cells were mixed in each case with 0.25 ml of the precipitate containing the DNAs. After incubation at room temperature for one hour, 2 ml of medium I were added to each, and the cells were incubated in a CO2 incubator at 39.5� C. for 2 hours (Kieny et al., supra). The ts N7 viruses originally used are unable to grow at this raised temperature, which permits selection of viruses which have undergone recombinations in the ts7 locus at least. Since the calcium phosphate lying on the cells had, in the long term, adverse effects on cell growth, the medium was removed after incubation for two hours, the cells were washed 3 times with 1 ml of PBS each time, agitating gently, the PBS solution was aspirated off each time, 2 ml of medium I was again added to the cells in each case, and the latter were kept in a CO2 incubator at 39.5� C. for a further 2 days. The culture dishes containing the cells and the medium were then frozen at -30� C., thawed again, the still adhering cells were scraped off the dishes, and the suspension was treated briefly with ultrasound as described above. For the second selection step, 0.2 ml of this homogenate, undiluted or diluted 1:5 or 1:30, respectively, with PBS, was placed in each case on an almost confluent lawn of human 143B TK- cells in 8 cm2 culture dishes from which the medium had previously been removed. The infected cells were maintained at room temperature for 1 hour and then a layer of 2 ml of agarose medium II (medium I plus non-essential amino acid solution [Gibco; Order No. 043-1140] plus vitamins [Gibco; Order No. 043-1120] plus 1% agarose) containing 0.1 mg/ml bromodeoxyuridine (BUdR) was placed on each. The dishes were then incubated in a CO2 incubator at 37� C. for 16-24 hours. A second layer of agarose medium II, but containing in addition 0.2% neutral red, was placed on top, and the dishes were incubated for a further 16-24 hours. After this time the colourless plaques were normally readily visible, and it was possible to remove the virus contained therein in form of an agarose cylinder, as a single clone by stabbing with a Pasteur pipette (plaque purification). Clones removed by stabbing in this way were grown on CVl cells as usual and, after growing, subjected to a second and third plaque purification. The final growing and purification of the recombinant WR 5.1 virus obtained in this way were carried out as described above.
As discussed above, and as set out in Table I, the MVA virus is, although no plaque formation occurs, able to grow in CVl and 143B TK- cells. This fact facilitates recombination of foreign antigens in the TK gene of the MVA virus in two ways. On the one hand, it has emerged from the experiments that CVl cells provide a better host than CEF for the recombination process. On the other hand, owing to the replication of the virus in 143B TK- cells, selection for TK- virus is also simplified.
The recombination of foreign antigens in the MVA virus differs, essentially in two respects, from the method described for the WR virus. On the one hand, it was not possible, in order not to alter the structure of the MVA virus except in the TK gene, to use selection via the temperature-sensitive virus ts N7. On the other hand, in the BUdR selection the 143B TK- cells were mixed not with agarose medium II but with normal medium II (medium I plus non-essential amino acid solution [Gibco, Order No. 043-1 140] plus vitamins [Gibco; Order No. 043-1120], and the removal of individual plaques by stabbing was not carried out until after infection of CEF with the selected TK- virus and incubation under agarose medium I.
The recombination protocol used for the MVA virus approximately corresponds to the method described by Mackett et al., (Proc. Natl. Acad. Sci. USA 79, 7415-7419 [1982]) for the WR virus. Medium was removed from CVl cells on 8 cm2 culture dishes at 80-90% confluence, a layer of 0.2 ml of an MVA virus suspension was placed on top of each (0.1 pfu/cell) and they were incubated at room temperature for 1 hour. Then 2 ml of medium I were pipetted to each of the infected cells, and the cells were incubated in a CO2 incubator at 37� C. for 2 hours. The following steps correspond to the method used for the WR virus (see above), except that no WR virus DNA was added for making the calcium phosphate precipitation, the subsequent incubation of the cells was carried out at 37� C., and a layer of 2 ml of medium II without agarose but with 0.1 mg/ml BUdR was placed on top of the 143B TK- cells. After incubation for 2 days, the culture dishes were briefly cooled to -30� C., the solution was thawed again, the remaining cells were scraped off the substrate, and cells and medium were homogenized by brief ultrasound treatment as described above. After removal of the medium from 8 cm2 culture dishes which were 80-90% overgrown with CEF, various dilutions of this homogenate were added to the cells (0.2 ml/dish) and the infection was maintained at room temperature for 1 hour. Then a layer of 2 ml of agarose medium I was placed on top of the CEF, which were incubated at 37� C. for 16-24 hours, and then a second layer of agarose medium I (2 ml) which additionally contained 0.2% neutral red was placed on top. After a further 16-24 hours, the colourless plaques were readily visible, and it was possible to remove the virus contained therein, together with the overlying agarose, by stabbing with a Pasteur pipette. The virus clone isolated in this way was grown on CVl cells and, to purify the virus clone, selection on 143B TK- cells and the subsequent steps were repeated 1-2 times (plaque purification). The final growing and purification of the recombinant MVA 5.1 virus obtained in this way were carried out in the manner described above.
In order to test the expression of the malaria 5.1 Ag by the recombinant WR 5.1 and MVA 5.1 viruses in CVl cells, CVl cells infected with recombinant virus were sedimented by brief centrifugation in a bench centrifuge (Hettich Mikrorapid K, obtainable from A. Hettich AG, Bach, Switzerland; max. speed for 3 minutes at 20� C.), and the sediment was washed twice with PBS, taken up in PBS and applied to a glass microscope slide and left to dry there. Another method comprised culturing CVl cells directly on a slide, infecting them with the virus, after 1-2 days removing incubation medium from the cells infected in this way by washing with PBS, and allowing them to dry on the glass slide at room temperature. To fix the cells, the slides were incubated in acetone at -30� C. for at least 1 hour and then left to dry at room temperature.
Rabbit anti-5.1 antiserum diluted in PBS was placed on the prepared slides in such a way that the cells were covered with a layer of liquid. The slides were incubated in a humidity chamber at 37� C. for 1 hour, then washed several times with PBS and, without allowing them to dry, the second antibody, labelled with fluorescein isothiocyanate (FITC) and diluted in PBS (goat anti-rabbit IgG, Nordic GAR/Ig/FITC, obtainable from Biogenzia Lemania SA, Lausanne, Switzerland), was added to the cells. After incubation in a humidity chamber at 37� C. for one hour, the slides were washed several times with PBS and left to dry thoroughly. It was then possible to test the cell preparations for fluorescence emission in a microscope under UV light.
In order to be able to compare the expression of the malaria 5.1 antigen by the recombinant MVA virus with that of the recombinant WR virus in vivo, the anti-5.1 antibody production in mice which had been immunized with either of these recombinant viruses was measured. For this purpose, Swiss albino mice were injected subcutaneously twice, at an interval of 2 weeks, either with the 5.1 recombinant of the WR virus (108 pfu/mousel) or the 5.1 recombinant of the greatly attenuated MVA virus (109 pfu/mouse). 2 weeks after the final injection, the tip of the tail was cut off and a little blood was aspirated into a heparinized capillary (10 μl mouse). The capillary was blown out into 90 μl of PBS, and the serum fraction was obtained from this solution by centrifugation. Various serial dilutions of the serum were tested in an ELISA (Voller et al., The enzyme-linked immunosorbent assay (ELISA), Dynatech [1979], obtainable from Dynatech Produkte AG, Embrach, Switzerland) for the amount of antibodies contained therein and directed against the malaria 5.1 antigen. For this purpose, multititre plates (for example Dynatech Produkte AG; Order No. 655001) were coated with 0.25 μg/well of recombinant 5.1 antigen isolated from E. coli (Hope et al., Nature 308, 191-194 [1984]). The results of a comparative experiment of this type are compiled in Table II.
TABLE II______________________________________Antibody productionanti-5.1 antigen ELISA titre______________________________________a) WR 5.1 (108 pfu/mouse) 1:15,000 1:31,000 1:30,000 1:22,800 1:30,000 1:8,000 WR WT 1:200b) MVA 5.1 (109 pfu/mouse) 1:6,000 1:6,500 1:8,000 1:6,700 1:7,100 1:6,000 MVA WT 1:250______________________________________
Groups of five 3-week old Swiss Albino mice were immunized as described above a) with the recombinant WR vaccinia virus (WR 5.1) or b) with the recombinant MVA vaccinia virus (MVA 5.1), and the titres of anti-5.1 antibody in the serum were determined by ELISA. In addition, as a control inoculation, groups of 2 mice were injected with the corresponding amount of the starting viruses (WR WT or MVA WT). Whereas the titres measured in the control sera were 1:200 (WR WT) and 1:250 (MVA WT), the values in the sera from the animals treated with the recombinant viruses were an average of a factor of 30 (MVA 5.1) or 120 (WR 5.1) above the value for the control sera.
In contrast to the starting virus (MVA WT) the recombinant MVA virus (MVA 5.1) forms plaques on CVl cells. Hence, it gave the impression that the behaviour of the recombinant MVA virus in cell cultures had altered slightly compared with the MVA starting virus. Since this alteration was not attributable to a contamination by other strains of virus, the suspicion arose that the virulence might have increased or the host-virus behaviour might have changed due to the recombination of the 5.1 antigen in the viral TK gene.
The slight alteration in the behaviour of the recombinant MVA virus in cell cultures, namely the formation of plaques on CVl cells, prompted the question of whether there had possibly been an increase in the virulence caused by the insertion of the foreign DNA into the viral thymidine kinase gene. One possible way of examining this question was to measure the neurovirulence of the viruses. For this purpose, the LD50 value was determined after intracerebral injection of the viruses into 1- to 2-day old mice. Firstly, the concentrations of all five tested viruses were adjusted to an initial concentration of 4�109 pfu/ml with PBS. Starting from these, 10-fold dilutions in PBS were prepared, down to a concentration of 4�102 pfu/ml. The viruses used were the WR virus (ATCC No. VR-119=WR WT), the recombinant WR 5.1 virus based thereon (WR 5.1), the Elstree virus (ATCC No. VR-862 LS WT), the MVA starting virus (MVA WT), and its 5.1 recombinant (MVA 5.1). 1- to 2-day old mice (Swiss albino) were divided into groups of 5 mice and, in each case, 2 such groups were left in the care of a mother mouse in a cage. 5 mice for each type of virus and virus concentration received injections of 10 μl of the virus suspension into the left half of the brain, and the behaviour of the animals was observed for a period of two weeks, or until they died. The LD50 value for a type of virus was calculated from the survival rate of the mice in the individual groups using the formula of Reed et al. (Amer. J. Hyg. 27, 493-497 [1938]). The results of these experiments are compiled in Table III. It emerges that no increase in the pathogenicity of the virus, at least with regard to the neurovirulence, has occurred due to the recombination process in the MVA virus, and that there is a large difference in the neurovirulence of the MVA viruses (MVA WT and MVA 5.1) compared with the other vaccinia viruses.
TABLE III______________________________________Neurovirulence in mice Log LD50______________________________________ WR WT = -8.7 WR 5.1 = -7.7 LS WT = -5.8 MVA WT = -1.6 MVA 5.1 = -1.5______________________________________
Many modifications and variations of this invention may be made without departing from its spirit and scope, as will become apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims.
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DENVER—DaVita announced that its annual charity bike ride, Tour DaVita, raised $900,000 to support Bridge of Life—DaVita Medical Missions in 2012.
The proceeds from Tour DaVita will help fund nine medical missions taking place in 2013 where Bridge of Life volunteers will be able to install or repair 112 dialysis machines. Through these missions, Bridge of Life will bring dialysis treatment to more than 600 people in communities that otherwise would not have access to this life-sustaining care.
"Tour DaVita participants collectively rode 70,000 miles, which means that for every four miles a rider completed Bridge of Life can provide a dialysis treatment to someone at one of our partnering clinics – someone's loved one will receive a second chance at life," said Lori Vaclavik, executive director for Bridge of Life—DaVita Medical Missions. "We are proud of all the riders and sponsors who made this gift possible. Because of them, people will live."
More than 470 participants each pedaled approximately 250 miles over the course of three days in Iowa Sept. 16-18. The ride kicked off in scenic Wilder Park in Allison, Iowa, continuing on through the rolling hills of the American Heartland – all in the effort to raise awareness and funds in the fight against CKD.
Since it started in 2007, Tour DaVita has helped raise nearly $5 million in the fight against kidney disease. Participants have collectively ridden 420,000 miles over the years, challenging themselves and their fellow riders to go as far as possible.
DaVita thanks the sponsors who made this gift of life possible, including ASD Healthcare, Cigna, Baxter, NxStage, Office Depot, Golden Construction, Henry Schein, Promotional Images, KPMG, Pentec Health, and Tata Consultancy Services.
Life-saving dialysis is not available in most of the developing world. Currently there are 112 countries that do not have resources for dialysis or kidney transplants, which directly results in the death of more than one million people a year from kidney failure. The Centers for Disease Control and Prevention recognize CKD as a major public health problem that not only reduces the quality and length of life, but is also expensive to treat.
Bridge of Life—DaVita Medical Missions an independent 501(c)(3) non-profit organization working to reach people who are disproportionately affected by CKD and live in developing countries abroad and disadvantaged communities in the U.S. Kidney disease is reaching epidemic proportions not only in the United States, but worldwide.
For more information about the 2012 Tour DaVita, please visit www.tourdavita.org.
For more information about Bridge of Life—DaVita Medical Missions, please visit www.bridgeoflifemm.org.


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Violin For Dummies Learner's Package
by For Dummies | Item #: 439807 | Model #: FDV-100 | (3) |
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About this item
This new addition to the ever-popular For Dummies line includes the violin and bow, case, music stand, tuner and Violin Basics book and CD.
Learn to play the violin in no time! The package includes a Maestro full-size violin and bow, Violin Basics For Dummies Book & Instructional CD, Digital tuner with LED readout, plush-lined hardshell case, hardwood shoulder rest, folding music stand and bag, extra fine tuners and E-strings, horsehair bow and rosin.
- Maestro full-size violin and bow
- Includes Violin Basics For Dummies Book & Instructional CD
- Digital tuner with LED readout
- Plush-lined hardshell case
- Hardwood shoulder rest
- Folding music stand and bag
- Extra fine tuners and E-strings
- Horsehair bow and rosin
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8 1/4" x 23 1/4" x 3 1/4"


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Boston, MA (PRWEB) October 07, 2012
The Doctors Health Press, a publisher of various natural health newsletters, books, and reports, including the popular online Doctors Health Press e-Bulletin, is reporting on a study that looked at antimonium tartaricum (AT). Actually a poison, AT can be an effective homeopathic remedy for relief from coughing and nausea.
As Doctors Health Press e-Bulletin notes (http://www.doctorshealthpress.com/general-health-2/a-poisonous-solution-to-coughs-and-nausea), AT was used way back in the 1500s to disinfect and clean open wounds. Unfortunately, nobody knew that toxins quickly entered the blood and this treatment often lead to fatal side effects. AT was also used to induce vomiting, but sometimes it didn't occur, and the poisons remained in the stomach.
As the Doctors Health Press e-Bulletin article, "A Poisonous Solution to Coughs and Nausea," outlines, to remind people not to fret about homeopathic remedies derived from toxic substances; just be sure to get them from a reputable source. They are carefully distilled and diluted and, by the toxins are long gone by the time it is taken as a remedy. Here, it is the pure compound tartar emetic that gets dissolved in water.
The article states what AT is best used for:
The article also notes who will benefit most from AT. Symptoms that indicate AT as a remedy often include a cold sweat, a lack of thirst, a white-coated tongue, and a pale face. Other symptoms may include sunken eyes and a blue color creeping across the lips. AT is often used for chest infections in older patients—especially if they are weak and if they present with a cough. Mental symptoms are not too strong, and thus are difficult to connect to AT. Adults can be anxious and apathetic, somewhat saddened by everything.
(SOURCE: Byun, W., et al., "Effect of positive health factors and all-cause mortality in men," Med. Sci. Sports Exerc. Sept. 2010; 42(9): 1,632–8.)
Doctors Health Press e-Bulletin is a daily e-letter providing natural health news with a focus on natural healing through foods, herbs and other breakthrough health alternative treatments. For more information on Doctors Health Press, visit http://www.doctorshealthpress.com.
The Doctors Health Press believes in the healing properties of various alternative remedies, including Traditional Chinese Medicine. To see a video outlining the Doctors Health Press' views on Traditional Chinese Medicine, visit http://www.doctorshealthpress.com/chinesemedicine.


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The race has been a part of the sport since the birth of the world championship in 1950, with Monza a mecca for motor sport fans.
Only once has the fabled venue failed to stage the race, in 1980 when it was switched to Imola, but it may now be axed altogether once the present agreement expires in 2016.
"It's not good," Ecclestone said when asked by Italian newspaper Gazzetta dello Sport about the future of the race. "I don't think we'll do another contract as the old one has been disastrous for us from a commercial point of view, so it's bye-bye after 2016."
Ferrari's circuit at Mugello has been mooted as a likely replacement, although Ecclestone claims he has not yet received any proposal. One key factor behind the race's potential departure is an alarming drop in F1's TV audience in Italy, with Ferrari's lack of form not helping.
"TV ratings are lower in Italy than elsewhere," Ecclestone added. "Channels have split with digital broadcasting, and there's also Twitter, Facebook and other communication means, so young people are more distracted.
"How to improve Italian ratings? If Ferrari started to end up first and second in qualifying and races, TV ratings would improve everywhere. Ferrari is a worldwide passion."
Meanwhile, Christian Horner, the Red Bull team principal, has praised Sebastian Vettel for his reaction to the team's ongoing crisis. Following an abysmal Austrian Grand Prix, Horner slated power-unit supplier Renault for their performance, describing it as unacceptable.
After suffering an electrical fault on lap two which almost resulted in Vettel grinding to a halt - he got going again but dropped to the back of the pack - Red Bull opted to retire their four-time world champion after 34 laps to save the car. It was the third time this year Vettel has been forced from a race, as many as incurred in the previous three years combined.
"He's been remarkably mature about it, to be honest with you," said Horner. "He's not been shallow in any way, he's not spat out his dummy or thrown the toys out, and he's had the majority of the problems this year."


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14 Things You Should Know About the Interview
Planning to wing your next interview? Not being fully prepared for an interview can be the biggest detriment in obtaining your job! Let us provide you the "need to know" understanding of the interview process and tips on how to conduct yourself and respond appropriately.
Sign up for this workshop to learn about:
- Understand the interview in the context of the job search process
- Understanding the selection of appropriate interview attire
- Common questions employers ask and tips on responding to questions effectively
- Behavior-based questions employers ask and how to prep for them
- Questions to ask the employer
- How to end the interview
- Interview follow-up
Contact Us if you have any questions
You might also be interested in these other workshops:
- Resume & Cover Letter: Market Yourself on Paper
- 14 Things You Should Know About the Interview
- 10 Steps to Making a Great Impression at the Career Fair
- Job Search Tips for New Graduates
- Thinking about Graduate School
- Applying for Graduate School
- Using LinkedIn to Network & Find Jobs
- U.S. Department of State Information Session
- CIA Information Session


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This new text from the editors of the classic Handbook of Empirical Social Work Practice draws upon the significant advances in research over the last several years. Featuring contributions from leading social work scholars, each chapter examines evidence-based approaches to the full spectrum of mental disorders as classified by the DSM.Edition/Copyright: 07
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For those who want a nice camera, try this one from MIUI:https://docs.google.com/folder/d/0B4huyVgvdssGdFozM3RlTWtqaTg/edit
Have you been able to find exposure settings is this? I had a look in it, and I found that the shutter button opens up zoom control, but the +/- on the left side looks to me like white balance, not exposure. Thanks