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Jack Harris : The Goose Is Out! , The Old Nun's Head, Friday 8 May.
The Old Nun's Head, 15 Nunhead Green, London SE15 3QQ.
7.45pm/ £7
Floorsingers welcome.
The Goose has (have?) admired Jack Harris for a long time as a fantastic singer and songwriter, and we thought it was about time we gave him a headlining gig!
Jack was the first international (ie non-American) artist to win the New Folk songwriting competition at the Kerrville Folk Festival, Texas. Previous winners include Steve Earle, Lyle Lovett, Gillian Welch and Devon Sproule.
Jack has gigged frequently in the UK, USA and Europe, both as a support act and headline performer. He has made several appearances at The Green Man Festival, and opened for some of folk music's biggest names, including Dick Gaughan, Martin Carthy and Dave Swarbrick, as well as contemporary songwriters like Anais Mitchell and Jeffrey Foucault.
Jack's live show is a riveting mix of song-craft and story-telling, adorned by intricate fingerstyle guitar and smoky, soulful vocals. Drawing from the narrative traditions of folk and blues music, Jack brings both into the here and now; a "born poet", whose songs are steeped in myth and mystery.
"Jack Harris is a priest of song who holds himself to a rigorous, ancient code of beauty most of us have forgotten exists."— Anaïs Mitchell
Here's Jack


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We are helping you to get your vote on this morning on Taking It to the Streets. The League of Women Voters is hosting a voter registration drive to help qualifying former felons be able to cast their ballots in November. Joining me this morning to tell us about the drive is Karen Kimball – with the League of Women Voters of Arlington, Virginia.
The League of Women Voters of Arlington is hosting a Voter Registration Drive for Qualifying Former Felons on July 23rd from 1pm to 3pm at Macedonia Baptist Church in Arlington. More information at: www.lwv-arlingtonva.org
Tune in to Taking it to the Streets Weekdays at 6:47, 7:13 and 8:13 on the Steve Harvey Morning show on 96.3 WHUR 96.3 FM
Follow me on Facebook and Twitter @bobbygailes for all updates and to stay connected.
Bobby Gailes
Follow Me: Facebook
Bobby is the producer of the syndicated Steve Harvey Morning Show heard weekday mornings from 6am – 10am. He can also be heard on the weekends as the host of the "Weekend Review" on WHUR 96.3 and on Sirius/XM Channel 141 and on WHUR-World. Bobby is also the host of "The New Artist Feature" also airing on Sirius/XM Channel 141.

subset: commoncrawl


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Sara Whitman is a senior business administration major with concentration in marketing and minoring in communication. She is from St. Charles, Missouri. Sara is a member of Phi Sigma Pi, a co-ed honors fraternity. She currently holds the position as Treasurer. Sara has also been involved in Phi Beta Lambda, a professional business organization open to all majors, where she held the position of Vice President of Philanthropy. She was also involved in the Tru-Leaders program sponsored by Boeing and is a part of the mentor program sponsored by Boeing this year as well. She also worked as a Student Advisor in Blanton-Nason-Brewer Residence Hall her sophomore year. Sara has interned at Technology Partners, Inc. in Chesterfield, Missouri in the Integrated Marketing and Communication Department and at Boeing in St. Louis, Missouri in the Supplier Management Department. She is looking forward to starting full time with Boeing after graduation as a member of the Business Career Foundation Program, a two year rotational program in the Finance Department. In her spare time, Sara enjoys hanging out with friends and family, playing tennis, and shopping.
Office Hours:
Monday: 1:30-2:30pm
Tuesday: 10:00-10:30am
Thursday: 10:00-10:30am, 2:30-4:00pm


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Thermaltake Armor A30 Case Review
To test the Thermaltake Armor A30 mATX case I will be taking some temperature readings on the APU and hard drives. These temps will then be compared against two other mATX cases to see how it holds up, and if the design makes the difference it needs. To simulate loads I will be running Prime95, small FFTs for the APU, and HD tune for the hard drive for one hour. I will be using AMD Overdrive for the APU and HD Tune for the hard drive, to monitor temperatures. All of the cases will be using their stock default fan setup that comes in the package. No additional fans will be added, keeping the tests fair. All of the components will also be run using their stock voltages, speeds, and latencies to keep anything from interfering with the scores.
Testing Setup:
- Processor: AMD Dual Core E350 APU
- Motherboard: Gigabyte Fusion E350
- Memory: 2x 2GB DDR3 2000MHz G.SKILL Flare @ 1066MHz
- Video Card: Integrated AMD Radeon HD 6130 Graphics Processor
- Power Supply: Ultra X2 750W Modular PSU
- Hard Drive: 1 x Seagate 750GB SATA
- Optical Drive: LG DVD+/-RW
- Case: Thermaltake Armor A30
- OS: Windows 7 Pro 64 bit
- Comparison Case #1: In Win Dragon Slayer mATX Case
- Comparison Case #2: Cooler Master Elite 341 mATX Case
Overall it was a close race, but the Thermaltake Armor A30 with the combined large fan and largely mesh design pulled off the lower temperatures by a hair.


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Hey Guys!
Figured Id make an account after viewing over cars for the last while now.
Im Brent, from, New South Wales, Australia, I drive a pretty stock for the moment
Lexus IS200
Plan on making a buildthread shortly! But heres a pic of it the day i got it
20170812_122317 by brent rushby, on Flickr
So yeah, hopefully things go well, always appreciate information that can be shared
Not the most mechanical minded, but am a, boilermaker by trade, so alot of the work
Ill be doing myself!


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Just a quick pop in to say HI! I have been so busy since Sunday that there has just been no time to blog. I have had a scrapping marathon, with all the deadlines being for today & tomorrow, I have been heads down in my paper, ink & glue. I did it though, I finished & sent off everything, eight 12x12 L.O's in 2.5 days....that has got to be my all time best achievement yet. I am so relieved it is all done & dusted tho', because now we can go away for the weekend & I can relax.
We are heading to a fishing camp near Bonnivale, which is just short 3 hours away (nice & close). The accommodation is right on the waters edge....& the dams are heavily stocked with fish...so I can lie in bed & chuck a line into the water from out of the window & be guaranteed to catch a large mouthed bass with hardly any effort at all.......my type of relaxation.
Where the kids will be at this point in my fishing fantasy, I have no idea....preferably in life jackets FAR away from the large fish I'll be landing. Oh....Pops.....! (my Mom isn't coming with us, she is still in Port Elizabeth, with my terminally ill Aunt :o( We will miss her not being with us, but we understand her needing to be with Aunty Col.
On a more cheerful note, my August Cocoa Daisy kit came....it is so beautiful! Have a squizz .....Aren't the colours amazing! This is the reason I have been MIA the last little while.
Well guys I am bushed. So I am going to have a LARGE cup of vanilla coffee, curl up on the couch with my delish bloke for a bit & then hit the sack.
Have a great day...or nite for those on my side of the equator.


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Passionate musicians are opening up the world of classical music to school kids in Mid Sussex.
Ensemble Reza, who have teamed up with West Sussex Music, are set to work with more than 300 children in an education project based on the BBC Ten Pieces initiative.
The BBC has selected ten classical pieces to encourage children to create responses to the works through music, digital art or dance.
Working with Ensemble Reza team schools will create their own music based on one of the Ten Pieces in a series of workshops. The students will then perform them at Ensemble Reza's Ten Pieces Music Festival Concert on Thursday, July 17, at the King's Church, Burgess Hill (7pm).
The project involves six primary schools: Holy Trinity, Cuckfield; Bolnore Village Primary School; Harlands Primary School, Haywards Heath; St Giles Primary School, Horsted Keynes; Blackthorns Community Primary School, Lindfield, and Northlands Wood Community Primary School, Haywards Heath, together with Oathall Community College, Lindfield.
"The pieces are the starting point, the original source of inspiration for the children," says Pavlos Carvalho, cellist for Ensemble Reza.
"We will listen to the piece together and start asking them to listen out for certain things or they'll tell us what they find interesting about the piece. It might be a rhythm. It might be a particular colour change in the music or it might be a particular feel. We take that particular rhythm and then see if the kids can create something of their own based on that.
"For example, if you were doing a Beethoven symphony – the fifth – they could take that and make something different just from the rhythm. It doesn't have to be those notes but it can be something that, if we heard it, even if it wasn't obvious, we would know that it's inspired by Beethoven's Fifth."
This isn't the first project Pavlos has worked on that aims to introduce classical music to kids. Last March Ensemble Reza, who formed in 2013, performed their first ever family concert at Holy Trinity Church, Cuckfield. Pavlos also worked with students from Blackthorns Primary School and Oathall Community College to help them create music for Romeo and Juliet.
He makes it clear why he considers this work important.
"First of all it's just beautiful music," he states. "Secondly, we live in a time where this particular classical music is often being sold in a cheap way to try to encourage students or young people to hear it so they don't actually hear what it's about."
Pavlos explains that people will often put a drumbeat on the music, add a video or simply distil the piece because they don't respect children's ability to concentrate on the work in its original form.
"Some children grow up never actually realising what a substantial and beautiful part of music they're missing," he continues. "Sometimes their fate can be determined for them simply because they're not exposed to something like this."
But this kind of project isn't just rewarding for children. Pavlos often finds the imaginative ideas that kids come up with to be very inspiring.
"As a trained musician one always tries to leave the imagination open," he says. "Sometimes education unlocks certain doors that can feed the imagination, but other times there are certain boundaries and limitations that one is aware of and rules that you wouldn't break."
But children approach music free of any rules, he explains. Without proper knowledge of concepts such as harmonic progression and melody, kids can sometimes suggest remarkable things. It's humbling, says Pavlos, and shows how even trained professionals have something to learn.
He offers an example: "The last project we did – the Romeo and Juliet one – the theme tune of the whole project with all these children (many of them very experienced musicians) came from a little girl who didn't play any instruments. She didn't even have singing lessons. She just went away after we talked about it and sat with me at the piano and sung me this idea, a series of three or four notes that she had in her head. From that we produced the most unbelievable, beautiful, emotional waltz and it came from a girl who's never done any music in her life."
For this project the Ensemble Reza musicians are working with the highly accomplished composer, presenter and musician Jason Rowland.
In the past twelve months Jason has led music projects for The Royal Opera House, The Philharmonia, The London Philharmonic Orchestra, The Royal Philharmonic Orchestra and Britten Sinfonia. He was also the musical director for the Canary Wharf Christmas sing-a-long concert.
And if that isn't enough, throughout June and July Ensemble Reza are also planning to create a Community Orchestra to rehearse every Wednesday evening. The group will be conducted by Pavlos Carvalho and will learn a selection of the Ten Pieces to perform at the Ten Pieces Music Festival Concert.
Ensemble Reza are making an application to Arts Council, England for funding towards the project. They are also looking for local sponsors.
To join Ensemble Reza's Ten Pieces Festival Community Orchestra, or to find out about supporting the Ten Pieces Project, contact firstname.lastname@example.com.
To find out more about the musicians visit ensemblereza.com.
Find out more about the BBC's Ten Pieces project here.
Pavlos Carvalho will perform a programme of great romantic cello music at Great Walstead School, Lindfield, on Saturday, February 28 (7pm). The show features cello sonatas by Frank and Rachmaninov. Tickets cost £13 for adults and £5 for children and students under the age of 18. See Carousel Music in Haywards Heath, Pepperbox in Cuckfield or SWALK in Lindfield.


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Philly Margarita March!
Saturday, April 30, 2016 at 12:00 PM - Sunday, May 1, 2016 at 10:00 PM (EDT)
Event Details
The Margarita March is an awesome margarita tasting tour at some of Philly's best venues. Join hundreds of fellow revelers as you enjoy a specialty margarita at each venue included in your ticket! Get ready for Cinco de Mayo in style.
Dress Code: Participants are encouraged to wear their favorite Cinco de Mayo gear, so put on a sombrero, poncho, or heck, your favorite outfit from that Spring Break Trip to Cancun...
How It Works:
- Check In between 12:00-3:00pm on April 30th or May 1 to pick up your Pass. No passes will be available for pickup after 3pm. Saturday passes can only be used Saturday. Sunday passes can only be used Sunday.
- After that, proceed in any direction and at any pace you choose!
- The Bartenders at each venue will punch a hole in your Pass to indicate you have visited there and been served their designated margarita
- The event concludes at 10:00pm.
This offer is final sale and nonrefundable. Event happens regardless of weather. Redemption of this offer is subject to normal availability. Pass is not shareable or transferable. Pass is valid on date of event only. Pass must be picked up between the designated check-in times otherwise forfeited. Types of margaritas are subject to change without notice depending on availability. Must be 21 or older to participate. Must agree to full liability waiver to partcipate. Price does not include tax and gratuity.
Any questions: Please email firstname.lastname@example.com.
When & Where
Fox & Hound
1501 Spruce St
Saturday, April 30, 2016 at 12:00 PM - Sunday, May 1, 2016 at 10:00 PM (EDT)
Add to my calendar


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association Insight Out (CD, £10.75)label: Now Sounds
This DELUXE EXPANDED MONO EDITION features the original mono mix of the album, which has been out of print since 1967. The 16-page, full-colour boo... more
paul revere & the raiders The Spirit of '67 (CD, £8.50)label: Now Sounds
The Spirit of 67 was fermented in the creative highs of 1966. Paul Revere & The Raiders were at the peak of their popularity that year, playing hu... more
cowsills On My Side (CD, £6.50)label: Now Sounds
The Cowsills possessed one of the most pleasing vocal blends in all of popular music. Released in 1971, On My Side was the band's swan song of their o... more
paul revere & the raiders Revolution! (CD, £9.75)label: Now Sounds
MONO MIX ON CD FOR THE FIRST TIME EVER! FEATURING 10 BONUS TRACKS! SOURCED FROM THE ORIGINAL MASTER TAPES! BILLBOARD TOP 25 LP! Released i... more
mc squared Tantalizing Colours: the Reprise Recordings (CD, £9.75)label: Now Sounds
Their Lost Reprise Recordings, 1967-1968! First Time Ever Released! From The Original Master Tapes! A Psych Pop Masterpiece Unleashed! MC SQ... more
harpers bizarre Anything Goes (CD, £9.75)label: Now Sounds
RARE MONO MIX! INCLUDES 9 BONUS TRACKS! FROM THE ORIGINAL MASTER TAPES! AN ESSENTIAL SUNSHINE POP CLASSIC! Anything Goes, HARPERS BIZARREs ... more
every mothers' son Come On Down: the Complete MGM Recordings (CD, £9.75)label: Now Sounds
BOTH OF THEIR CLASSIC LPs on 1 CD! FIRST TIME EVER COMPILED ON CD! FROM THE ORIGINAL MASTER TAPES! EVERY MOTHERS SONbest known for their Summ... more
paul parrish The Forest of My Mind (CD, £6.75)label: Now Sounds
Sounding like an American Donovan, PAUL PARRISH burst onto the scene in 1968 with this ambitiously produced gem of a record filled with psychedeli... more
ruthann friedman The Complete Constant Companion Sessions (CD, £6.75)label: Now Sounds
Produced in 1969 by JOE WISSERT (The Turtles, Lovin Spoonful, Jackie DeShannon) Constant Companion is Ruthanns only LP during the first chapter ... more
cryan' shames A Scratch In the Sky (CD, £6.75)label: Now Sounds
The Cryan Shames second album, 1967's A Scratch In The Sky, is an unsung gem of that heady musical era. The sets distinctive mix of youthful ga... more
donna loren These Are the Good Times: the Complete Capitol Recordings (CD, £6.75)label: Now Sounds
A regular vocalist on ABC-TVs Shindig!, and cast member of American International Pictures Beach Party series, DONNA LOREN was a recording artis... more
tiny tim God Bless Tiny Tim (26-track CD, £6.75)label: Now Sounds
Born of that brief, inspired moment in time when the freaks almost took over, April 1968s God Bless Tiny Tim is an otherworldly mission statement f... more
book a trip 2: more psych pop sounds of capitol records (24-track CD, £6.75)label: Now Sounds
Once more, NOW SOUNDS raids the Capitol Records vaults in Hollywood for late-60s Psychedelic Pop/Soft Pop obscurities and unearths a treasure trove... more
serendipity singers Love Is a State of Mind (22-track CD, £6.75)label: Now Sounds
ESSENTIAL SUNSHINE POP FROM 1968! FIRST TIME ON CD ANYWHERE! FROM THE ORIGINAL MASTER TAPES! INCLUDES BOTH STEREO & MONO LP MIXES! As the fo... more
association The Association (22-track CD, £9.50)label: Now Sounds
Never known as an album band, with the release of The Association, this band proved to listeners that it was moving in the right direction. Helped alo... more
ruthann friedman Windy: a Ruthann Friedman Songbook (CD, £6.75)label: Now Sounds
Having written The Associations Windy while living in David Crosbys basement, RUTHANN FRIEDMAN remained an intriguing and mysterious figure of 60... more
sugar shoppe The Sugar Shoppe (CD, £6.75)label: Now Sounds
A lovely title on CD! This popular Toronto band released several singles during 1967-'68 and this self-titled debut album for Capitol in 1968. The gro... more
del shannon Home & Away (CD, £6.75)label: Now Sounds
Recorded at Londons Olympic Studios in February 1967, Home & Away was intended by Rolling Stones producer/Immediate Records founder Andrew Loog Old... more
mamas and the papas People Like Us (CD, £6.75)label: Now Sounds
DELUXE EXPANDED EDITION. THEIR 1971 SWAN SONG. FROM THE ORIGINAL MASTER TAPES! A reunion project, PEOPLE LIKE US was the final collaborative e... more
knack Time Waits for No One: the Complete Recordings (13-track CD, £6.75)label: Now Sounds
FIRST TIME EVER ON CD ORIGINAL RECORDINGS FROM 1966 & 1967 FEATURING FRANK ZAPPA ON SOFTLY, SOFTLY! FROM THE ORIGINAL MASTER TAPES! ATTENT... more


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What kind of vehicles do you have in your fleet?
We have almost anything imaginable from classic stretched limousines, SUV Limos, Hummer Limo, Party Buses, Ultimate Party Buses, Sedans, Roll Royces, Carriages and much more. If you are looking for a particular vehicle please do not hesitate to contact us.
What happens if I cancel?
There is usually a 20% non-refundable deposit upon reserving your vehicle. This fee is for holding the date of your vehicle and will not be refunded.
How old do you need to be able to rent a limo?
We require an adult 21 years or older to sign the limo service agreement.
How far will you travel?
He travel all over the tri-state area and our Coach buses will go where ever you want to!
What are the limousines stocked with?
Most vehicles will come stocked with ice, water and soda. You are more than welcome to bring your own drinks as well.


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Don't forget the humble cuttle fish this coming month
What is it that us Brits have against cuttlefish? This forgotten creature is now in season – for a limited time only – but are you going to see it on supermarket fish counters or on menus up and down the land? Hardly. Despite being plentiful in British waters, delicious and comparatively sustainable, we just don't seem to eat it over here. Instead we ship tons of it to places like Spain, Portugal and Greece, where they appreciate its merits and it will command a far higher price.
But if you like deep fried calamari or squid risotto, there's no reason why this "poor relation" shouldn't be just as good – or even better. Tougher and more meaty than squid, it either needs to be cooked very quickly (or served raw, as they do in Japan) or long and slowly until extremely tender. Anywhere in between and it will be like chewing on a rubber band.
These varied cooking methods make it incredibly versatile, so it can be used in a whole host of dishes. I like it sliced thinly and soaked in milk, dusted in flour, deep fried and dipped in garlic mayonnaise. It's equally good quickly pan-fried with lemon and paprika (or, even better sumac, a North African spice with a citrus tang), and served with crusty bread and tomatoes for a starter or light lunch.
It's also great in a Spanish-style stew, cooked for hours in white wine, garlic and olive oil which almost pickles it and means it will keep for ages. For a radical twist on an old favourite, one of my favourite chefs, Pierre Koffmann has an amazing cuttlefish bolognese recipe.
If you're going to find it in this country, your best bet is to try a specialist fishmonger or a fish market where you'll pick it up far cheaper than squid (but get them to prepare it for you – it can be messy) or in oriental or Mediterranean restaurants. Although there are British chefs who do use it when it comes into season, it would be great to see it become more widely recognised as one of our great national fish. It's tasty, healthy, abundant, and inexpensive. What's not to like – give it a go. You've got until the end of June.


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Shakespeare's Birthplace Trust
Harvard HouseHigh StreetStratford-upon-AvonWarwickshireCV37 6AU
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Tiger-Sul has announced a new Nutrient Calculator app that the company says will help farmers more accurately plan for fertilizer applications and meet their soil's nutrient needs.
The app contains several features. Among them is the Nutrient Removal Calculator, where users start by selecting among many common row crops, including corn, soybeans, wheat, canola, cotton, rice, sorghum and peanuts. Next, enter in yield goals and the app estimates how much nutrients will be removed from the soil. It covers all macro, secondary and micronutrients, including N, P, K, S, Mg, Ca, Cu, Mn, Zn, B and Fe.
On the app's ROI calculator, select between corn or wheat and use sliders to change grain price and price of Tiger-Sul's TIGER-90CR sulfur product. That can help users calculate whether they can expect a positive return on investment from using that product.
Users also have access to an image library that shows what nutrient deficiencies look like in most major row crops.
The Nutrient Calculator app is free to download for both Apple and Android devices. For 99 more farming app reviews, go to www.AgWebAppFinder.com.


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Share your opinion on what the Qur'an says about Apostates. Join the discussion.
'Kill Him Who Changes His Religion'
As everyone knows, Islam is not based solely on the Qur'an. There are also hadiths [verified sayings of the Prophet Muhammad], which support the death penalty for apostasy.
Ibn Abbas, relates the Prophet said, "Kill him who changes his religion, or "behead him".
Narrated Anas: Some people from the tribe of Uki came to the Prophet and embraced Islam. The climate of Medina did not suit them, so the Prophet ordered them to go to the(heard of milch)camels of charity to drink their milk and urine(as medicine). They did so, and after they had reverted from their ailment they turned to renegades (they reverted from Islam)and killed the shepherd of the camels and took the camels away, The Prophet sent some people in pursuit and so they were caught and brought, and the Prophet ordered that their hands and legs should be cut off and their eyes should be branded with heated pieces of iron, and that their cut hands and legs should not be cauterized, till the die.
Reason For Concern
The Qur'an can be pretty scary all on its own.
But any culture that elevates belief, in the absence of evidence, to the highest place in the hierarchy of human virtues is a culture to be concerned about.


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COSTA MESA — It had been 21 years since Estancia High last won an outright football league title.
The Eagles were in a rush Friday night to end the drought.
Estancia scored its first three touchdowns in the Battle for the Bell fast and put away Costa Mesa, 35-6, at Jim Scott Stadium, closing out an undefeated Orange Coast League season.
The Eagles ended a two-game losing streak in the rivalry game and hurt the Mustangs' CIF Southern Section Southern Division playoff hopes.
When the postseason begins next week, Estancia (6-4, 5-0 in league) will have a home game. The Mustangs' playoff fate was out of their hands. They needed to upset the Eagles to keep their chances alive.
Costa Mesa (3-7, 2-3) will miss the playoffs after Calvary Chapel defeated Godinez, 47-35. Calvary Chapel and Godinez each finished 3-2 in league and earned the league's second and third playoff entries.


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The Bolivia Mission Initiative of the Susquehanna Conference is partnering with its counterpart in the New York Conference to form a United Methodist Volunteers in Mission Team to work with our brothers and sisters of the Bolivia Methodist Church Central District. You are invited to be part of this exciting opportunity to spread God's love.
Encounter with Christ in Bolivia - Cochabamba, Bolivia
Trip Planned Each Year late June through early July (length of trip can vary from 14 to 17 days)
Cost: Expected range of $2,500 to $3,000 (depending upon airline prices, length of stay and inflation in Bolivia)
Total cost includes: Airfare to/from Bolivia, visa, in-country transport, room & board, other
mission activities.
It does NOT include: Passport cost, inoculations, transport between home & departure airport,
personal costs
Deposit Required: $300 (The deposit holds your place and is refundable until March 25)
If you have any questions, contact the Bolivia Mission Coordinators:
VIM Bolivia 2012 Trip Report #1 – Sunday Evening, June 24
This is the first report of the 12-member VIM Bolivia 2012 Team, a small but mighty band of loving servants from Connecticut (Rachel Bird, Rev. Bill Pfohl, Cindy Svensen), New York (Dennis Angle, Chris Freeman), Pennsylvania (Diane Hetrick, Kathy Silva, Ginny Stevenson, Mary Thrush), New Hampshire (John Borchert, Rod Wendt) and even Minnesota (Anders Svensen).
For 3 of us, this is our first Bolivia VIM experience.
Summary: we arrived Friday safely and without incident; Saturday got right to work on construction work and Vacation Bible School preparation; Sunday
worshipped in THREE churches; and are gelling into an amazing Team!
The Bolivia Mission Initiative
Has declared that the month of May 2012 will be known as
During this month, please Make a Joyful Noise as often as possible and help us to
For the people of the Methodist Church of Bolivia Central District (Cochabamba and Cotani)
How can we Make A Joyful Noise?
It only takes a few simple steps:
• Designate one or more weeks (or the entire month) as your congregation's JOYFUL NOISE time and advertise the event to
• Get some large metal pots and label them saying "Please make a Joyful Noise and Change the World for disadvantaged
people in Bolivia" and "Drop Your Loose Change Here".
• Let people know that the Joyful Noise of the coins dropping in the pots is the laughter of Bolivian children. (Also let them
know that currency and check are welcome—the immediate noise they make dropping into the pot is more muted, but the
children's laughter is just as real.)
• Display these pots around the church and/or circulate them through the congregation during worship immediately after your
regular offering. (Letting your children and youth pass the pots is a great way to get them involved in this mission.)
• Send a check for the total collected, payable to Susquehanna Conference and designated for Conference Advance # 63654-
Bolivia Mission Initiative, to Bob & Ginny Stevenson, Bolivia Mission Coordinators, 714 Ash Court, Lebanon, PA 17042 or to
the Susquehanna Conference Treasurer. Unless indicated otherwise with your submission, funds will be split where needed
most between the School Supplies and Mustard Seed programs described below.
How will this Change the World?
For the Children
• Funds are used to purchase school supplies for children in Methodist Church daycare, Sunday-
School and after-school mentoring programs.
10 years ago, poor Cochabamba families considered themselves fortunate to have a "family pencil"—one writing instrument shared by all the children striving to learn to write, as well as their parent(s). When the first Encounter with Christ Volunteers in Mission team arrived in 2002 and led a VBS, Ginny Stevenson put a white paper and crayon in front of a young girl. The girl simply stared, having no idea what to do with these items, until Ginny marked a spot on the paper. What followed was the dawning of whole new world for this girl, who spent hours happily filling the paper with "pictures" only she could produce. Each of the 15 Encounter VIM teams going to Bolivia since 2002 collected from congregations here at home and carried with it school supplies and other materials for use by Cochabamba's children. However, this year the airlines are charging a fee of $70 for each suitcase of these items the team might carry to Bolivia. Faced with this excessive cost,
the Team will not collect school supplies here at home but is requesting contributions of funds that can be used to purchase these important items in Bolivia.
For all the People of the Central District
• Funds are used for the Encounter with Christ Mustard Seed Program, to start or expand self-help programs that will improve
the economic and social situation for members of Methodist Churches and surrounding communities.
Funding for this program has grown from about $600 in 2002 to approximately $4,000 in recent years. Recently funds
were used to:
- Support three indigenous young adults from Cochabamba Methodist Churches studying at seminary in Argentina.
- Purchase of playground and nursery equipment for daycare programs at two of the Churches.
- Support the evangelism, children's and women's programs in the newly organized and rapidly growing Luz de Vida
(Light of Life) congregation.
Programs to be supported in 2011 will be determined during the VIM Team's trip (June 21 – July 7) and depend upon
Central District needs and fund raised.
For more information about this program or other aspects of the Bolivia Mission Initiative, please contact Bob & Ginny Stevenson at


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I should have started a blog long ago. I've made a million excuses..
"I don't have the time."
"I don't even know what I would write about."
"Who's gonna read it, anyway?"
Today, I decided to stop making excuses.
I can make the time.
I'll write about whatever I want until I develop my niche'.
Who cares if anyone reads it. At least I'll be writing SOMETHING.
So, here I am, a few hours after making that decision, writing away..
I've enjoyed reading for ages. I used to spend hours lost in a book, so absorbed in the world that the words created, that I'd often forego food and sleep because I just couldn't put it down. Through my love for reading, I gained tremendous respect for the power of the written word, and the writers who created it.
My favorite books growing up were fantasy books. I loved and respected how the authors could create, not only a story, but an entirely new world to base that story in. I give full credit to the Harry Potter series for creating my love for reading.. I got to grow up alongside the main character. I have no shame in admitting that I waited for my letter from Hogwarts. As an adult, I still harbor a love for fantasy books, but I've learned to expand my horizons.
I tried writing some stories when I was a teenager, and I really enjoyed it, but I just didn't have that natural creative spark, and I found myself constantly frustrated of that fact. I'm an analytical thinker; very left-brained, and, with time, I realized that I was a fairly decent writer, but my real niche' was in editing the hell out of any piece of writing you put in front of me. When writing, I would get a paragraph or two into a story, then spend 15 minutes going through and making sure each and every sentence sounded perfect. I couldn't help myself. This never turned out to be a good idea because by the time I tried to get back to writing the story, any flow I had was long gone. I just couldn't move on until I was satisfied.
When I got to college, I still wanted to get into writing, any kind of writing, but I also wanted to be sure I got into a field that had decent job potential. After a couple years of slowly completing some of my core classes, while I figured out what I wanted to do, it hit me. Journalism.
Admittedly, when I first got into my journalism classes, I was in it strictly for the writing, not the journalistic aspect. Looking back, that was pretty naive since journalism involves a lot more aspects than just writing. I learned that lesson quickly after writing a few articles of my own. As time went on, and I took more classes, I got more and more excited about journalism as a whole. My primary excitement stemmed from the passion of some of my professors about how journalism can help to expose wrongdoings, and create good changes in our world. Being able to make a difference through my writing was a very appealing prospect to me.
Now, as I get further along in my college career, I've begun to lean more towards wanting to get into an editing position of some sort. Not just because I'm good at it, but because I really enjoy it too. That being said, I still love writing. And I absolutely love the idea of being a writer. The respect and love I gained for writers at a young age has never dissipated, and it still drives me today. Admittedly, the writing process can be hard, but my love for the written word continues inspiring me to push forward.
There's a great quote by writer/poet Dorothy Parker that says, "I hate writing; I love having written." Now, I don't hate writing, but the process can be extremely frustrating. Yet, when I see the finished project of my writing, no matter the content, I love it. I love having written. I love to see the story, or impression, my words can create, and that, well.. that makes it all worth it.
Several different people have given me the same advice when it comes to writing, especially when I talk about not knowing where to start…
"Just write."
Just write.
It's high time I took that advice.
I may not be writing the short stories, or novels, that I long to create one day, but the more I write, the better I'll become, and the happier I'll be.. just having written.


text:
8 Mar 2011: Létourneau S, Im EJ, Mashishi T, Brereton C, Bridgeman A, et al. (2011) Correction: Design and Pre-Clinical Evaluation of a Universal HIV-1 Vaccine. PLOS ONE 6(3): 10.1371/annotation/fca26a4f-42c1-4772-a19e-aa9d96c4eeb2. https://doi.org/10.1371/annotation/fca26a4f-42c1-4772-a19e-aa9d96c4eeb2 View correction
One of the big roadblocks in development of HIV-1/AIDS vaccines is the enormous diversity of HIV-1, which could limit the value of any HIV-1 vaccine candidate currently under test.
Methodology and Findings
To address the HIV-1 variation, we designed a novel T cell immunogen, designated HIVCONSV, by assembling the 14 most conserved regions of the HIV-1 proteome into one chimaeric protein. Each segment is a consensus sequence from one of the four major HIV-1 clades A, B, C and D, which alternate to ensure equal clade coverage. The gene coding for the HIVCONSV protein was inserted into the three most studied vaccine vectors, plasmid DNA, human adenovirus serotype 5 and modified vaccine virus Ankara (MVA), and induced HIV-1-specific T cell responses in mice. We also demonstrated that these conserved regions prime CD8+ and CD4+ T cell to highly conserved epitopes in humans and that these epitopes, although usually subdominant, generate memory T cells in patients during natural HIV-1 infection.
Therefore, this vaccine approach provides an attractive and testable alternative for overcoming the HIV-1 variability, while focusing T cell responses on regions of the virus that are less likely to mutate and escape. Furthermore, this approach has merit in the simplicity of design and delivery, requiring only a single immunogen to provide extensive coverage of global HIV-1 population diversity.
Citation: Létourneau S, Im E-J, Mashishi T, Brereton C, Bridgeman A, Yang H, et al. (2007) Design and Pre-Clinical Evaluation of a Universal HIV-1 Vaccine. PLoS ONE 2(10): e984. https://doi.org/10.1371/journal.pone.0000984
Academic Editor: Douglas Nixon, University of California at San Francisco, United States of America
Received: September 5, 2007; Accepted: September 13, 2007; Published: October 3, 2007
Copyright: © 2007 Létourneau et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: TH is the Jenner Institute Investigator. The work was supported by MRC UK.
Competing interests: The authors have declared that no competing interests exist.
Despite twenty-five years of global effort, an effective vaccine against the human immunodeficiency virus type 1 (HIV-1) remains elusive. Induction of broadly neutralizing antibodies against HIV-1 is very difficult, yet it is the key to all other protective anti-viral vaccines[1]. Therefore consideration of HIV-1 vaccine candidates that stimulate cellular immunity has been the focus of many recent vaccines[2]. Although recent advances in vector design have generated optimism in this field[3], [4], these technologies still need to address the extreme variability of HIV-1, whereby co-circulating viruses may differ in over 20% of their proteome[5], [6]. Thus, while novel vectors and heterologous prime-boost combinations are getting better at inducing higher frequencies of HIV-1-specific T cells, less attention has been paid to how these vaccines can elicit T cells capable of recognizing multiple HIV-1 variants.
There are several approaches for dealing with the HIV-1 diversity. One optimistic view is that a single clade may induce sufficiently cross-reactive T-cell responses to protect against other variants of both the same and heterologous clades. The choice of a natural isolate can be based on having the closest sequence to all others, or picking a strain derived from acute infection and arguing that there is a convergence of viral sequences during transmission[7]. However, even if a single variant elicits responses that confer some cross-reactive protection, such protection is likely to be only partial and thus it is well worth attempting to design vaccine immunogens with enhanced cross-reactive potential. Although there are numerous reports of cross-clade reactive HIV-1-specific CD8+ T cell responses[8]–[12], use of unphysiologically high concentrations of variant peptides make the biological relevance of many of these results uncertain. In contrast, there are ample examples of highly specific T cell receptors sensitive to single amino acid (aa) changes[13]–[19], as well as compelling evidence of HIV-1 variants escaping existing T cell responses in infected individuals by single mutations in epitopes[2], [20]–[22]. In vitro, systematic studies employing all possible single aa substitutions in each position of an MHC class I epitope indicated that as few as one third of such epitope variants were recognized by a given T cell receptor[13], [19]. These results are in agreement with theoretical predictions proposed for cross-recognition of MHC class I-presented peptides by T cell receptors[23]. Thus the use of a single natural isolate for a vaccine has a high risk of not protecting against a different clade, nor against many variants of the same clade.
A second approach to HIV-1 diversity derives vaccine immunogens from 'centralized' sequences, which employ consensus/average, or centre-of-the-tree[24] sequences or extrapolated aa to a common clade or group ancestor[5]. Centralized sequences are designed to minimize the sequence differences between a vaccine immunogen and circulating viruses[5], [24]–[26]. So far they have proven immunogenic and able to elicit T cell responses in small animal studies[24], [27]–[29] and clinical trials[30]–[32], providing experimental support for their further development. Early results for centralized immunogens for the entire group M are promising in that initial immunogenicity studies in mice yielded T-cell responses that were comparable to within-clade responses for many clades[29], however, this strategy may be stretched too far for optimal coverage of CD8+ T cell epitope variants of the whole group M[18], [19], [33].
In a third approach, vaccines deliver a cocktail of immunogens derived from different clades[34]–[36]. While initial results have been encouraging and responses to each antigen in the cocktail were observed[36], attention still needs be paid to possible immune interference, such as epitope antagonism, between different, but closely related peptide sequences in the vaccine, which may be limiting responses to some epitopes. Antagonism of T cell responses by altered epitope peptide ligands has been demonstrated both in vitro[37], [38] and in vivo[18], [39]–[42]. It can occur when a host capable of mounting a response to an agonist epitope is simultaneously exposed to an antagonist epitope variant, which interferes with the induction of the T cell response to the agonist epitope and leads to a defective response. Thus, the breadth of responses induced by cocktail approaches should be carefully monitored when such vaccines are used[18].
A fourth approach uses computational methods for assembling a polyvalent vaccine candidate that optimize the coverage of T cell epitopes. 'Mosaic' immunogens[43] are based on intact proteins and retain the probability for natural processing and presentation of T cell epitopes. Their potential problems are similar to those of other cocktails of natural proteins, i.e. immune interference and inclusion of both variable and conserved regions, whereby responses to variable regions may draw attention away from potentially more useful conserved targets. The impact of these processes will be only resolved in vaccine studies. An alternative means of designing immunogens contending with the HIV-1 variation is the COT+ method[44], which combines a central sequence with a set protein fragments designed to help cover diversity.
Here, we describe a further alternative that may have considerable advantages. We describe the construction and experimental testing in mice and humans of a novel multi-clade immunogen derived only from highly conserved regions of the HIV-1 consensus proteome, which was designed to provide extensive coverage of the principle HIV-1 clades A, B, C and D, while minimizing the possible occurrence of the epitope interference. It has the potential considerable advantage of focusing the T cell responses on the most conserved parts of the virus and thus overcoming the usual patterns of immunodominance, while making it difficult for the virus to escape without a likely significant cost to its fitness. This approach has also merit in the simplicity of design and delivery, requiring only a single immunogen to provide extensive coverage of global HIV-1 population diversity.
Preparation of the pTH.HIVCONSV and pTH.HIVCONSVdH DNA vaccines
The synthetic gene coding for HIVCONSV (GeneArt) was subcloned into plasmid pTH [45] and the codons coding for the H epitope were deleted using a PCR assembly to generate pTH.HIVCONSVdH. The plasmid DNA for immunizations was prepared using the Endo-Free Gigaprep (Qiagen) and stored at −20°C until use.
Preparation of the AdHu5.HIVCONSV and AdHu5.HIVCONSVdH vaccines
Recombinant adenoviruses were obtained using the AdEasy™ Adenoviral Vector System (Stratagene), following the manufacturer's instructions. Both adenoviruses expressed the green fluorescent protein as a marker.
Preparation of the MVA.HIVCONSV and MVA.HIVCONSVdH vaccines
Recombinant MVAs (rMVAs) were made as described previously [46]. Briefly, chicken embryo fibroblast (CEF) cells grown in Dulbeco's Modified Eagle's Medium supplemented with 10% FBS, penicillin/streptomycin and glutamine (DMEM 10) were infected with parental MVA at MOI 1 and transfected using Superfectin (Qiagen) with 3 µg of pSC11.HIVCONSV or pSC11.HIVCONSVdH carrying also the β-galactosidase gene as a marker. Two days later, the total virus was harvested and used to re-infect CEF cells. rMVAs were subjected to five rounds of plaque purification, after which a master virus stock was grown, purified on a 36% sucrose cushion, titered and stored at –80°C until use.
Six-well tissue culture plates containing sterile microscope coverslips treated with 0.01% poly-L-lysine (Sigma) were seeded with 1×105 cells/well of HEK293T. After 12–24 h, cells reached 80% confluency and were transfected with 5 µg of a selected plasmid using the Superfectin (Qiagen), according to the manufacturer's recommendations, or infected with a selected recombinant viral vector at MOI 1 or 10. Cells were then incubated for 18–24 h, fixed with 0.5% formaldehyde, washed with PBS, their membranes perforated with 50% ethanol for 5 min, 70% ethanol for 10 min, and 50% ethanol for 3 min. Cells were washed again and blocked overnight with 2% FCS in PBS (FCS/PBS) at 4°C. The FCS/PBS solution was replaced by a 1/200 working dilution of a primary mouse monoclonal antibody against the Pk tag (Serotec) in FCS/PBS and incubated for 3–18 h at 4°C. Cells were subsequently washed three times with PBS, and incubated with a 1/500 dilution of the Alexa fluor 594-conjugated secondary chicken anti-mouse monoclonal antibody (Molecular Probes) in FCS/PBS for 2 h at room temperature or for 3–18 h at 4°C. Alternatively, a FITC-conjugated mouse monoclonal antibody against the Pk tag was used at a 1/500 dilution in FCS/PBS, and incubated for 2 h at room temperature or 3–18 h at 4°C. The cells were then washed three times with PBS, mounted on a microscope slide with Vectashield DAPI nuclear stain (Vector Laboratories) and photographed on a Zeiss fluorescence microscope.
For all animal experiments, groups of four 5- to 8-week-old female BALB/c or HHD mice were used. All animal procedures and care were approved by a local Ethical Committee and strictly conformed to the UK Home Office Guidelines.
Mouse immunizations and preparation of splenocytes
Under general anaesthesia, mice were immunized i.m. with 100 µg of pTH.HIVCONSV DNA, or 106 PFU of AdHu5.HIVCONSV or 106 PFU of MVA.HIVCONSV. On the day of sacrifice, spleens were removed and pressed individually through a cell strainer (Falcon) using a 5-ml syringe rubber plunger. Following the removal of rbc with Rbc Lysis Buffer (Sigma), splenocytes were washed and resuspended in Lymphocyte Medium [R-10 (RPMI 1640 supplemented with 10% FCS, penicillin/ streptomycin), 20 mM HEPES and 15 mM 2-mercaptoethanol] at concentration of 2×107 cells/ml.
Peptides and peptide pools
HPLC-purified, overlapping 15- by 11-mer peptides spanning the entire HIVCONSV protein were obtained from Sigma Genosys. Peptides were at least 80% pure as verified by mass spectrometry. Individual peptides corresponding to epitopes were synthesised in an in-house facility (Weatherall Institute of Molecular Medicine, Oxford). All peptides were dissolved in DMSO (Sigma) at a concentration of 50 mg/ml, and stored at −80°C.
Murine intracellular cytokine staining
Two million splenocytes were added to each well of a 96-well round-bottomed plate (Falcon) and pulsed with peptides or peptide pools and kept at 37°C, 5% CO2 for 90 min, followed by the addition of GolgiStop™ (BD Biosciences). After a further 5-h incubation, reaction was terminated, the cells washed with FACS wash buffer (PBS, 2% FCS, 0.01% Azide) and blocked with anti-CD16/32 (BD Biosciences) at 4°C for 30 min. All subsequent antibody stains were performed using the same conditions. Cells were then washed and stained with anti-CD8-PerCP or anti-CD4-PerCP (BD Biosciences), washed again and permeablized using the Cytofix/Cytoperm™ kit (BD Biosciences). Perm/Wash buffer (BD Biosciences) was used to wash cells before staining with anti-IL-2-FITC, anti-TNF-α-PE and anti-IFN-γ-APC (BD Biosciences). Cells were fixed with CellFIX™ (BD) and stored at 4°C until analysis.
Murine ELISPOT assay
The ELISPOT assay was performed using the Becton Dickinson IFN-γ ELISPOT kit according to the manufacturer's instructions. The membranes of the ELISPOT plates (BD Immunospot™ ELISPOT Plates) were coated with purified anti-mouse IFN-γ antibody diluted in PBS to a final concentration of 5 µg/ml at 4 °C overnight, washed once in R-10, and blocked for 2 h with R-10. A total of 2.5×105 splenocytes were added to each well, stimulated with or without peptide for 16 h at 37°C, 5% CO2 and lysed by incubating 2x with deionized water for 5 min. Wells were then washed 3x with PBS 0.05% Tween-20, incubated for 2 h with a biotinylated anti-IFN-γ antibody diluted in PBS 2% FCS to a final concentration of 2 µg/ml, washed 3x in PBS 0.005% Tween-20 and incubated with 50 mg/ml horseradish peroxidase-conjugated to avidin in PBS 2% FCS. Wells were washed 4x with PBS 0.005% Tween-20 and 2x with PBS before incubating with an AEC substrate solution [3-amino-9-ethyl-carbazole (Sigma) dissolved at 10 mg/ml in Dimethyl formaldehyde and diluted to 0.333 mg/ml in 0.1 M acetate solution (148 ml 0.2 M acetic acid and 352 ml 0.2 M sodium acetate in 1 liter pH 5.0) with 0.005% H2O2]. After 5–10 min, the plates were washed with tap water, dried and the resulting spots counted using an ELISPOT reader (Autoimmune Diagnostika GmbH).
51Chromium-release assay
Isolated mouse splenocytes were restimulated in vitro with 2 µg/ml of peptide in the Lymphocyte Medium for 5 days, at 37°C 5% CO2. On day 5 the cells were washed three times in R-0 and diluted two-fold in a 96-well U-bottom plate (Nunc) to yield the different effector to target ratios. ELA A2-Kd or JK A2-Kd target cells were labelled with 51Chromium at 37°C 5% CO2 for 90 min with or without appropriate peptide, washed three times with R-0, added to the effector cells at 5×103 target cells per well, and incubated at 37°C 5% CO2 for 4–6 h. The percentage of peptide-specific lysis was calculated as [(sample release-spontaneous release)/(total release-spontaneous release)]×100. The spontaneous release was less than 10% of the maximum release.
Human PBMC samples
For healthy lab subjects, PBMC separation was performed within 2 h of blood receipt. Blood was layered onto Ficoll (Sigma- Aldrich) and centrifuged (40 min, 400 g, without brake) at room temperature. Following centrifugation, the cellular interface was removed, diluted in Hanks buffer (Sigma-Aldrich), and re-centrifuged. Cryopreserved PBMC samples from vaccine clinical trial participants in Oxford and African patients were used for detection of HIV-1-specific effectors. Cells were washed once more with 50 ml RPMI (Sigma-Aldrich) and then suspended in 10 ml RPMI for counting. Cells were counted using a Coulter Z1 Counter (Beckman-Coulter). Trypan blue exclusion (Sigma-Aldrich) was used to estimate the percentage of viable cells. All studies involving human samples were approved by local Ethical Review Panels and all patients gave an informed consent for donation of blood samples.
Short-term culture of PBMC
Short-term cell lines were set up as described previously[31]. Briefly, on day 0, fresh or frozen PBMCs were washed three times in R-0 and resuspended at 2×106 cells/ml in RAB-10 (RPMI 1640, 10% human AB serum) supplemented with 25 ng/ml of IL-7 (R&D Systems). Twelve-well tissue culture plates (Nunc) were seeded with 0.9 ml of the PBMC suspension. Hundred ml of either Pool 1-3, Pool 4-6, or the FEC Pool (positive control of Flu-EBV-CMV CD8 epitopes) was added to each well for a final concentration of 1.5 µg/ml for each individual peptide. On day 3 and 7, 1800 IU of IL-2 (Chiron) were added to each well, as well as 1 ml of RAB-10 on day 7. On day 10, cells are washed twice in PBS, resuspended in fresh RAB-10 and rested for 26 to 30 h. Cells were subsequently tested for IFN-γ production in an ELISPOT assay with or without CD8+ cell depletion as described below.
CD8+ cell depletion
CD8+ cells were depleted using the Dynabead Depletion Kit (Dynal) following the manufacturer's recommendation. Briefly, PBMCs were resuspended in a small volume with biotinylated anti-CD8 monoclonal antibody (BD Biosciences), incubated for 20 min at 4°C, the excess antibody was then washed off, cells were incubated with Dynabeads for 20 min at room temperature on an orbital shaker, and separated by a magnet. The depleted cells were collected and washed twice.
IFN-γ ELISPOT assay
PVDF membrane ELISPOT 96-well plates (Nunc) were pre-wet by dispensing 50 µl of 70% ethanol, incubating at room temperature for 5 min and washing three times with endotoxin free PBS (Sigma). Plates were then coated overnight at 4°C with 50 µl of a 10 µg/ml purified anti-human IFN-γ antibody solution in PBS. Plates were blocked with R-10 for 2 h, 80,000 cells/wells in 50 µl of R-10 were added to each well (40,000 cells for FEC lines), as well as the appropriate peptide pool in 50 µl of R-0 (final concentration of 1.5 µg/ml or each individual peptide) or relevant controls from pre-aliquoted peptide plates. Plates were incubated at 37°C, 5% CO2 for 14 to 18 h. Wells were then washed six times with PBS 0.05% tween-20 and incubated for 2 h with a biotinylated anti-IFN-γ antibody diluted in PBS 0.5% BSA to a final concentration of 1 µg/ml. Wells were washed again six times with PBS 0.05% tween-20 and incubated for 1 h with a horseradish peroxidase complex (Vector Laboratories) in PBS. Wells were washed four times with PBS 0.05% tween-20, two times with PBS, and incubated for 4 min with an AEC substrate solution (3-amino-9-ethyl-carbazole (Sigma) dissolved at 10 mg/ml in dimethyl formaldehyde and diluted to 0.333 mg/ml in 0.1 M acetate buffer [148 ml 0.2 M acetic acid and 352 ml 0.2 M sodium acetate in 1l of distilled water, pH 5] with 0.005% H2O2). Wells were finally washed three to five times with tap water to stop the reaction, dried, and spots were counted on an ELISPOT reader (Autoimmune Diagnostika). Results were expressed as Spot Forming Units per million cells (SFU/106 cells). Responses were considered positive if they were four times higher than background (no peptide) and if the background was less than 100 SFU/106 cells.
Statistical analysis
An unpaired student t-test was used to determine significant difference between the averages of mock-stimulated splenocytes and splenocytes restimulated with a particular peptide in ICS assays in BALB/c mice, and between the averages of mock-stimulated splenocytes and splenocytes restimulated with a particular peptide pool in IFN-γ ELISPOT assays in HHD mice, PBMC unpulsed and peptide-pulsed, and was performed using the program available at http://www.physics.csbsju.edu/stats/t-test.html. Responses were defined as positive if p<0.05.
Design of the HIVCONSV immunogen
A novel immunogen, termed HIVCONSV (for conserved), was designed as a chimaeric protein and assembled from the most highly conserved domains among the HIV-1 clade A, B, C and D proteomes. First, a decision was taken that the HIVCONSV gene should be approximately 2.5 kbp in size, which makes it suitable for most currently used genetic vaccine vectors and is likely to support a high protein expression. Two and a half thousand nucleotides translated into fourteen, 27- to 128-aa-long, most conserved regions of the HIV-1 proteins (Fig. 1A). The centralized sequence method was employed and the HIVCONSV immunogen was assembled from segments derived from one of the four within-clade consensus sequences (Fig. 1B) to reflect the fact that even the most conserved regions of HIV-1 are somewhat variable. It should be noted that because these regions are so highly conserved, often the consensus for one clade perfectly matched the consensus sequences of the other clades or indeed the group M (Fig. 1C), enhancing the potential for eliciting globally relevant cross-reactive responses. To keep the vaccine simple and minimize occurrence of immune interference of T cell responses while ensuring a good coverage of all the four major clades, we alternated the clades of individual segments in the 'string' (Fig. 1C and D). Epitopes recognized by rhesus macaque and mouse CD8+ T cells, and a mAb[47]–[49] were added to the C-terminus of the HIVCONSV immunogen (Fig. 1B–D), to facilitate the vaccine pre-clinical development.
(A) Localization of the 14 most highly conserved regions of the HIV-1 proteome. The numbers written vertically under each fragment boundary indicate the first and last aa positions using the HXB2 reference strain numbering (http://www.hiv.lanl.gov/content/hiv-db/LOCATE/locate.html). (B) Predicted aa sequence of the the HIVCONSV immunogen with indicated fragment numbers. (C) Summary of the fragments including: the fragment number; the protein in which it was embedded; the clade of the consensus sequence selected for inclusion in the immunogen, alternating between clades A–D; additional clades that have identical HIVCONSV; and position numbers in the chimeric vaccine. The number of additional clades with identical consensus sequences to selected clade reflects the high level of conservation in these regions, and is encouraging in terms of the global potential of the vaccine. The consensus sequences compared were to the M group consensus, clades A–K, and three very common recombinant circulating forms CRF01 (common in Asia and Africa), CRF02 (common Africa), CRF08 (common in China) retrieved from the Los Alamos database 2004 consensus alignment (http://www.hiv.lanl.gov/content/hiv-db/CONSENSUS/M_GROUP/Consensus.html). (D) Schematic representation of the HIVCONSV immunogen (not drawn to scale) indicating clade anternation (above), overlapping peptide pool derivation and protein origin by colour coding. (E) Hamming distances between the HIVCONSV antigen fragments and the global circulating viral sequences. The full M group alignment, including recombinant sequences, was used for the comparison. The Los Alamos database alignment contains only one sequence person, and contains sequences from between 600 and 1000 individuals in these proteins. The Hamming distance range for 95% of the sequences relative to the vaccine immunogen is given by the vertical lines. The distances between the full length natural proteins were then calculated relative to HXB2 reference strain Env, Vif, Gag and Pol sequences for comparison. Distance measures are minimal estimates, as gaps inserted in regions where insertions and deletions occur were not counted. (F) Numbers of known CD8+ T cell epitopes (defined to within 12 aa or less in the Los Alamos HIV-1 database) in each of the 14 conserved protein fragments included in the HIVCONSV immunogen are shown. When more than one HLA class I presenting molecules can present the same HIV-1 epitope, then each is counted as a distinct epitope; if more than one sequence variant has been described as an epitope presented by the same class I molecule, then these are counted as a distinct epitopes; however, if an HLA serotype and genotype that are potentially the same are each described as presenting the same epitope (like A2 and A*0201) they are scored as a single epitope.
Alignments of the HIVCONSV immunogen with the global HIV-1 sequences of group M including recombinant forms revealed that at least half of the sequences in the Los Alamos database are identical to fragments 6 and 8 (the median distance = 0), while fragments 2, 3, 10, 11, and 12 differ in less than 3% of their aa positions when compared to half of the sequences (median <0.03). The largest distance from the circulating global sequences displayed fragment 9 with differences in just over 7% aa positions.
Conserved HIV-1 protein regions were included into the HIVCONSV immunogen irrespective of whether of not they contained known T cell epitopes, however, every conserved fragment in the HIVCONSV contains at least one known human epitope (Fig. 1F). In fact, 270 (24%) of the 1112 distinct published CD8+ T cell epitopes smaller than 12 aa described in the Los Alamos HIV-1 database are embedded in these fragments. Even though most epitopes in the literature have been defined using clade B reagents and the HIVCONSV immunogen is an assemblage of HIV-1 clade A, B, C, and D consensus fragments, still 192 (71%) of these 270 HIVCONSV epitopes are identical to an experimentally defined epitopes and additional 59 (22%) differ by only one aa, so that 251 (93%) epitopes differ by no more than a single-aa difference from a known epitope and thus may elicit a cross-reactive response.
Vaccine construction and basic immunogenicity
The HIVCONSV gene was made synthetically using 'humanized' aa codons[50] and its open-reading frame was preceded by a consensus Kozak sequence to -12 nucleotides[51] to maximize protein expression. A Met start codon was added to the first fragment. For initial studies, the gene was inserted into plasmid pTH DNA, human adenovirus serotype 5 (AdHu5) and modified vaccinia virus Ankara (MVA) vectors described previously[52] to yield pTH.HIVCONSV, AdHu5.HIVCONSV and MVA.HIVCONSV vaccines, respectively. The HIVCONSV protein expression was demonstrated by immunofluorescence of transiently transfected or infected human embryonic kidney cells 293T using the mAb C-terminal tag (Fig. 2A–D). Using immunodominant epitope H, also known as P18-I10[49] and restricted by H-2Dd and Ld, the immunogenicities of individual vaccines and their heterologous prime-boost combinations were confirmed in the BALB/c mice. This demonstrated a strong priming with the pTH.HIVCONSV DNA and ability to boost responses by heterologous vaccine vectors measured by ex vivo interferon (IFN)-γ ELISPOT (Fig. 2E) and 51Cr-release (not shown) assays. Particularly strong responses were elicited by the pTH DNA-rAdHu5-rMVA regimen (further designated DAM) delivering HIVCONSV reaching a mean of 3375 spot-forming units (SFU)/106 of freshly isolated splenocytes.
A histochemical and DAPI staining of 293T cells transiently transfected with pTH.HIVCONSV DNA (A), or infected with MVA.HIVCONSV (B) or AdHu5.HIVCONSV (C and D). HIVCONSV protein expression was detected using mAb tag Pk at the C-terminus of the immunogen and a primary anti-Pk mAb followed by secondary FITC- (A and B) or AlexaFluor584- (C and D) conjugated detection antibodies. The AdHu5.HIVCONSV vaccine also expressed GFP, which co-localized with the HIVCONSV expression (D). (E) BALB/c mice were immunized using the regimen indicated below, and the HIVCONSV-induced T cell responses were assessed in an ELISPOT assay using the H epitope. Results are shown as a mean±SD (n = 4). U–unimmunized; D–pTH.HIVCONSV DNA; A–AdHu5.HIVCONSV; and M–MVA.HIVCONSV. For doses and timing, see Methods.
Breadth of HIVCONSV vaccine-induced immune responses in the BALB/c mice
The HIVCONSV chimaeric protein is not a natural protein, and the new context resulting from concatenating the fragments may impact the processing of intact epitopes that are embedded within the fragments. It was therefore important to demonstrate that HIVCONSV can induce T cell responses in mice and that epitopes recognized by human HLA-restricted T cells can be generated. We noted, as previously, that the response to the added H epitope dominated the T cell response in BALB/c mice (Fig. 3A). To avoid the H epitope domination of the T cell responses in the BALB/c mice[18], [53], vaccines expressing HIVCONSVdH immunogen with the H epitope deleted were constructed. Groups of BALB/c mice were immunized using the two strongest heterologous regimens from the previous experiment and the breadth of induced T cells was assessed using six pools of 32 peptides (15-mer overlapping by 11 amino-acid residues) corresponding to the whole HIVCONSV protein (Fig. 1D). While weak responses were observed following the DM regimen, higher frequencies of T cells recognizing at least 5 peptide pools were elicited by the DAM regimen (Fig. 3A, left and middle panels). In both instances, responses to pools 1–4 were dominated by the H epitope in pool 6 and were much stronger when the HIVCONSVdH immunogen was used (Fig. 3A, right panel). Next, we showed that the DM regimen of the HIVCONSVdH vaccines induced both CD8+ and CD4+ T cells, which could produce IFN-γ and IL-2 in response to antigenic stimuli (Fig. 3B). Following identification of the individual pool peptides, minimal previously identified peptides were confirmed for some responses minimal epitopes[53] (Fig. 3C). Further analysis demonstrated elicitation of high quality T cells capable of production of IFN-γ, IL-2 and TNF-α (Fig. 3D) and killing of targets sensitized with MHC class I-restricted peptides (not shown). Finally, using the HIVCONSV vaccines and the one CD4+ and five CD8+ T-cell epitopes, various dual and triple heterologous regimens were directly compared. This indicated that at the doses used, triple schedules were more immunogenic than the dual ones and indicated the superiority of DAM (Fig. 3E).
Mice were immunized using the regimen and immunogen indicated above (A, B and C) or below (D) the graphs and the HIVCONSV-specific responses were determined in ex vivo ELISPOT (A and E) or ICS (B and D) assays detecting the indicated cytokines and using for restimulation overlapping peptide pools schematically shown in Fig. 1D (A and B) or individual epitope peptides (D and E). (C) Identified peptides or epitope sequences and their origin, name and T cell reactivity. In (D): white–IFN-γ; black–IL-2; stripy-IFN-γ+IL-2; and grey–TNF-α; *-responses significantly above the no-peptide background (p<0.05). In (E): white–no peptide followed from left to right by epitopes H, G1, G2, P1, P2 and P3. Results are shown as a mean±SD (n = 4). For doses and timing, see Methods.
HIVCONSV vaccine-induced HLA-A2-restricted responses in transgenic mice
As a prelude to experiments in humans, we used genetically modified mouse strain HHD, which expresses as the only MHC class I molecule chimaeric human (α-1 and α-2) and mouse (α-3) HLA-A*0201 heavy chain covalently linked to the human β2m light chain. We used the most potent DAM regimen of the HIVCONSV vaccines for induction of HLA-A*0201-restricted T cells and detected responses in ex vivo IFN-γ ELISPOT assay recognizing two peptide pools (Fig. 4A). Using the same assay, the fine specificities of these responses were mapped to two previously described[54], [55] although relatively uncommon, minimal epitopes (Fig. 4B), which were confirmed in a 51Cr-release assay on murine and human target cell lines expressing the HLA-A*0201 molecule. The epitopes were fully conserved in three out of the four consensus clade sequences, but the one aa substitution of the one outlier clade did not affect the killing (Fig. 4C). Note that the VIYQYMDDLY epitope encompasses the reverse transcription active site.
(A) Mice were immunized using the DAM regimen and the vaccine-induced responses were detected in an ex vivo ELISPOT assay. Results are shown as a mean±SD (n = 4). For doses and timing, see Methods. (B) Identified epitope peptides and their origin. (C) Killing of murine EL4 A2-Kd (top) and human JK A2-Kd (bottom) target cells sensitized with the shown peptides in a 51Cr-release assay after a 5-day in vitro peptide re-stimulation. Black, grey and white bars indicated effector to target ratios of 100, 50 and 25 to 1, respectively.
Generation of HIVCONSV-specific responses in natural HIV-1 infection
Next, we demonstrated that natural HIV-1 infection generally leads to generation of HIVCONSV-specific T cell responses, although these are usually smaller than the commonly seen immunodominant responses to epitopes in the more variable regions of HIV-1 and required in vitro expansions. All blood donors were tissue typed and where possible, the clade of their virus was identified (Table 1). In order to maximize sensitivity, PBMCs from infected patients were expanded in culture in the presence of HIVCONSV-derived peptide pools prior to the IFN-γ ELISPOT assay as described previously[31]. In this highly sensitive assay, 0 of 9 healthy HIV-1/2-uninfected lab volunteers had detectable responses to HIVCONSV peptides, while 13 of 13 HIV-1-infected patients had HIVCONSV-specific memory T cells (Fig. 5A). In all five tested patients for whom we had sufficient frozen PBMC, depletion of CD8+ cells demonstrated that these responses were mediated by CD8+ T cells (Fig. 5B). These responses were broad and the median magnitude of all the HIV-1-infected patients for each peptide pool ranged between 1,000 and 3,500 SFU per 106 cells in the cultured IFN-γ ELISPOT assay (Fig. 5C). With the exception of patient 020, twelve HIV-1-infected individuals responded to at least two peptide pools and 6 to three or more (Fig. 5D). Thus, conserved regions of the HIV-1 proteome included in the HIVCONSV immunogen served as a source of T cell epitopes immunogenic during the course of natural HIV-1 infection.
The HIVCONSV-specific memory T cells were assessed in healthy and HIV-1-infected subjects using an IFN-γ ELISPOT assay after a 10-day peptide and cytokine culture. (A) Summed frequencies of HIVCONSV-specific cells detected in healthy (n = 9) and HIV-1-infected (n = 13) subjects. The bars show the group medians of 578 SFU/106 and 8,092 SFU/106 cells for the healthy and infected subjects, respectively. (B) In five subjects indicated below, cultured PBMC were left undepleted (grey) or depleted of CD8+ cells (black) prior to the ELISPOT assay. The difference between undepleted (median = 8,092 SFU/106 cells) and CD8-depleted samples (median = 550 SFU/106 cells) was statistically significant (p = 0.0313). (C) Responses to individual HIVCONSV-derived peptide pools as shown in Fig. 1D determined for the HIV-1-infected (grey) and healthy (black) subjects shown as medians. (D) Responses to individual peptides pools for each HIV-1-infected patient indicated below. Bars show a mean±SD of three assay wells and '*' indicates a positive response according to criteria set in Methods. Due to sample shortage, subject 021 was not tested.
A critical and contentious issue in HIV-1 vaccinology is how the vaccine-induced T cell responses will cope with both the intra- and inter-clade virus variabilities. Here, we describe a design and pre-clinical evaluation of vaccine immunogen HIVCONSV, which is based on the 14 most conserved regions of the HIV-1 proteome. These regions are well populated with known though less dominant CD8+ T cell epitopes, which are highly conserved. We demonstrated in BALB/c and HLA-A*0201-transgenic mouse strains that this vaccine immunogen can serve as a source of immunogenic epitopes. Furthermore, we detected HIV-1-specific memory T cells that could recognize HIVCONSV-derived peptides in 13/13 HIV-1-infected patients proving that HIVCONSV -specific responses are commonly generated during natural HIV-1 infection.
Pathogens such as HIV-1 with highly variable genomes still have relatively conserved regions of the proteome, which are structurally or functionally important. Some of these regions might reflect a lack of a selective immunological pressure, i.e. absence of immunodominant or any epitopes that select out escape mutations, although one would expect to see some background synonymous and non-synonymous variability if there were no fitness cost to substitutions in such regions. Here, we confirm both in mice and humans that the conserved HIVCONSV regions are not generally immunologically inert and contain T cell epitopes that are often recognized, although these tend to be less immunodominant than more variable epitopes in natural infection. This subdominance may be an advantage for the vaccine strategy, because it would allow elicitation of a T cell response that is different from that normally stimulated by HIV-1 (which fails to control the virus). The subdominance of many HIVCONSV epitopes and influence of immunodominance was clearly demonstrated in the series of BALB/c mouse immunizations using the HIVCONSV and HIVCONSVdH immunogens where a deletion of a single dominant 'H' epitope tripled the frequencies to several previously subdominant epitopes. Although 37 HLA-A2-restricted epitopes have been described, most patients only respond to the same two or three of these, which are immunodominant, but absent from the HIVCONSV immunogen [56]. Therefore, it was encouraging that the HLA-A*0201-transgenic mice nevertheless generated responses to 2 conserved subdominant epitopes, one of which include the reverse transcriptase active site. It is noteworthy that responses to some subdominant epitopes have been shown to be more protective than that those recognizing their immunodominant companions [57]–[59]. In natural HIV-1 infection, many of the conserved epitopes present in this construct are subdominant as implied by the need for expansion of patients' PBMC prior to the response detection. In contrast the more variable escapable epitopes dominate to the extent that HLA type imprints changes in virus sequence in the patient populations [60], [61]. Our approach offers the possibility of changing the natural immunodominance by pre-infection vaccination, focusing the responses on highly conserved epitopes more like those seen by long term non-progressors with HLA B27 or B57 (ref. [62]).
In this work, we also directly compared various heterologous two- or three-component vaccination regimens and found that the most potent at the vaccine doses used was a combination of the three employed vectors in a DNA priming followed by sequential boosts with rHAdV-5 and rMVA, or the DAM regimen. Although this may depend of the doses used and be immunogen specific[63], triple prime-boost regimens were superior to the dual deliveries and merit testing in clinical trials.
A theoretical problem with this vaccine construct, a consequence of the chimaeric nature of the immunogen, is the possibility that unnatural stretches of aa at the boundaries of the fragments could also elicit T cell responses. In the work here, we did not identify any response to a junctional epitope, although we cannot exclude this happening with some HLA types and when the vaccine is tested in humans these responses should be sought to ensure there are no cases of immunodominance weakening the true anti-HIV-1 responses.
Although T cell epitopes in conserved regions of HIV-1 proteins were identified previously[64]–[72] and their value for vaccine development has been recognized[71], [72], the HIVCONSV immunogen has a number of unique features. The HIVCONSV immunogen is a chimaeric protein assembled from protein regions rather than epitopes, which enables broader coverage overlapping epitopes presented by multiple HLA proteins; it uses consensus sequences of the four major HIV-1 clades, which are in many segments identical to consensus sequences from multiple clades and this should allow a geographically broad deployment of the HIVCONSV vaccines; it employs artificial clade consensus sequences designed to deal with the intra-clade variability; it combines sequences of the four clades sequentially rather than in parallel, which avoids epitope antagonism; and through the lower frequency of immunodominant epitopes, it favours induction of broader T cell responses. Our initial results indicate that human T cell responses to epitopes embedded in the chimaeric immunogen are common, although further study is needed to indicate how generalizable this is to other epitopes of HIVCONSV, and if the junction regions between fragments can stimulate problematic responses in humans. Finally, the theoretical global epitope coverage by the HIVCONSV immunogen is narrower compared to the considerably more complex "mosaic" gene strategy, but the balance between optimizing epitope coverage and vaccine simplicity perhaps favours our HIVCONSV approach.
The desirable, but not critical non-human primates studies with the HIVCONSV immunogen are underway. Although the advantages and disadvantages of the HIVCONSV vaccines or any of the other methods addressing the HIV-1 diversity discussed above can be argued theoretically and in model situations, whether or not any of them can induce broad enough anti-HIV-1 responses to decrease the transmission and/or reduce virus load in HIV-1-infected vaccine recipients will be only proven in clinical trials.
Author Contributions
Conceived and designed the experiments: TH AM. Performed the experiments: SL EI TM CB. Analyzed the data: TH BK SL. Contributed reagents/materials/analysis tools: TH LD TD AB HY. Wrote the paper: TH BK AM.
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text:
Did Jesus Compete with Satan to Become Savior of the World?
Jesus, Not Joseph, is the Reason for the Season
This broadcast will focus on the irrational veneration of Joseph Smith that occurs in the Mormon Church that is equal to, or over that of the Lord Jesus Christ, during the month of December. As Smith was born on December 23, 1805, frequently the focus of the Mormon is redirected to this false prophet, rather than to our only Prophet Jesus Christ.
Continue reading "CHRISTMAS – Jesus, Not Joseph, is the Reason for the Season"


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Clone a project on Codeplex to your own Codeplex repo with Mercurial.Published on Mar 02, 2010
If you use Github (Git) or Bitbucket (Mercurial) you are familiar with the ability to fork a project into your own repository in those sites.
Once forked you can start working in your own repo and once you are ready to integrate you will send a pull request to the master repository.
Codeplex recently started using Mercurial but it doesn't have (or I couldn't find) a way to clone a project directly from inside Codeplex.
UPDATED March 5: Yesterday Codeplex added support for forks when using mercurial.
First, cloning to your local computer
You need to have TortoiseHg installed on your computer before going forward with this tutorial.
First you need to check out the project you want to fork using the clone url:
Go to the source control page of the project and click on the source control connection instructions.
Copy the clone url and open the TortoiseHg clone window.
Enter the repository url as the source path and select a folder where to clone the repository. If you don't have a folder already created for this repo, just add the name in the destination path text field and the folder will be create.
Second create a project on Codeplex and add as a Sync source.
Log into Codeplex (or create an account) and create a project where you want to work on, make sure you select Mercurial as your source control.
You should be able to also use Google code to host your clone, everything should work the same, just make sure you use Mercurial as well.
Once the project is created go to your new project source control page and copy the address to your repository.
In your computer open the properties of your cloned repository (the one we cloned in the previous step).
Go to the sync tab and click in the Add button.
Notice that the default (an only) repository is the original one, we want to add the repo to our newly created project and set it as default.
If you are using https authentication make sure you add your username and password following this form: https://username:email@example.com/repository
Add any Alias name but not "default", not now. Click Ok.
Select the Remote repository marked as default and click the edit button, change the alias from default to "origin".
Now in the sync dialog select the new repository (the one pointing to your project) and click the Set as default button.
Once done you should have something like this, the name of the projects should reflect the projects you are working with.
At this moment you are ready to push the code to your new project, just open the synchronize window from TortoiseHg and click push.
Once this is done, go to the source code tab in Codeplex and take a look, notice that you should have all commits from the original project plus any new commits done into your clone, in this case I added three commits after cloning.
From inside Visual Studio
Or course this works from inside Visual Studio as well if you have the Hg plug-in installed