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I am delighted to welcome Jessica Redland to Jaffareadstoo
💮Jessica, welcome to Jaffareadstoo. Tell us a little about yourself and how you got started as an author.
My name’s Jessica Redland and I live in Scarborough, North Yorkshire, with my husband, our 14-year-old daughter and a 5-year-old sprocker spaniel called Ella who still thinks she’s a puppy. I’ve nearly always worked in HR, specialising in recruitment and training, but fulfilled my dream of becoming a full-time author last summer.
I’ve always loved reading and English was my favourite subject at school but I’d never thought about writing until I was in my late twenties. My manager at work said my business reports read more like stories and joked that I should write a book. I loved that suggestion but had no ideas for a plot. It was obviously meant to be because something happened to me that felt like the perfect premise and, once I started thinking about that idea in terms of a book, lots more ideas came to mind.
It took me a whopping ten years to write my first book! During that time, I had several major life-events like moving four times, opening and closing a shop, getting married, having a baby, and changing job several times so there were significant chunks of time when I wasn’t writing but I was always thinking about it. I also studied the craft as being good at writing and being good at writing a book are different skills.
I initially secured a publishing deal but the publisher ceased trading after only 18 months which was a blow. I got my rights back and re-released my titles as an indie author but struggled to make an impact so, after a few years, decided to try for another publishing deal. I was fortunate enough to be taken on by Boldwood Books in 2019 and have released edited versions of my backlist through them alongside several new titles.
💮For readers who aren’t familiar with your writing, what can they expect from your novels?
My books are currently set in a fictional seaside town in North Yorkshire called Whitsborough Bay or in Hedgehog Hollow – a hedgehog rescue centre in the Yorkshire Wolds countryside.
There is always a romance in my stories but it isn’t always the main focus of the story as I love to explore friendship, family and community too. My stories are uplifting and will usually raise some smiles and even a few laughs, but can be emotional too so some readers may reach for the tissues. I love my characters and will always guarantee them a happy ending.
💮Do you write the type of books you like to read and which authors have influenced you?
Very much so. When I started to write, I was hugely inspired by Jill Mansell and Marian Keyes. I’d read most of their books and I saw myself as an author of romantic comedies.
My writing developed over time and, across subsequent books, I found myself tackling more emotional subjects so, even though there may be some moments of humour, this isn’t something that’s consistent across the books so I wouldn’t call myself a romcom author anymore.
I love to read both types of romantic book and have discovered some amazing new-to-me authors through Boldwood Books as I try to read books from as many of my fellow Boldwood authors as I can.
💮Where do your ideas for your stories come from, and as one book finishes do you already know where the next book will take you?
My ideas come from all over the place. The inspiration for my debut novel – New Beginnings at Seaside Blooms – came from something that happened to me. I was at a crossroads in my career and my personal life and a friend gave me a gift voucher for a telephone clairvoyant. It’s not really my thing but I decided to give her a call and she told me I was going to move back home, open a shop, and I’d meet the man of my dreams called Steven. I didn’t find my Steven but I did find the plot for a book!
I gave my main character, Sarah, two best friends as I wanted one to support the clairvoyant’s predication and the other to rubbish it. They developed and it became apparent they had stories of their own so I had a trilogy on my hands. Then my original publisher asked me to write a short story linked to the series and I ended up writing a novella. I felt there was more story to tell so I wrote a short novel sequel to that and those two books combined to make the first book in the ‘Welcome to Whitsborough Bay’ series called Making Wishes at Bay View.
Seaside Blooms is on a cobbled street in Whitsborough Bay called Castle Street and I had ideas for all sorts of businesses on that street. The shop I ran in real-life (which I had been thinking about before the clairvoyant call) was a specialist teddy bear shop and I used that for partial inspiration for All You Need Is Love. I positioned that shop – Bear With Me – on Castle Street opposite Seaside Blooms.
I wanted to write a Christmas story and Castle Street seemed like a great setting for the season as it has old Victorian-style lamp posts and cobbles. Lovely. I wrote Christmas Wishes at the Chocolate Shop and mentioned another shop in that story – Carly’s Cupcakes. Suddenly I knew who Carly was and she had a story to tell. And so did her friend Tara who ran The Chocolate Pot – the café next door – so Christmas at Carly’s Cupcakes and Starry Skies Over The Chocolate Pot Café were born.
The premise for The Secret to Happiness came from a song lyric. The line was, “Wish I could tell you goodbye” and the song was about someone in a bad relationship who couldn’t let go and wanted her boyfriend to let go of her first. I started thinking about what scenarios there may be where someone needed to let go of someone or something and was struggling to do so. I came up with about six and narrowed it down to three very different ones. I wanted the power of friendship to be the trigger to say the goodbyes but it would have been too much of a stretch for three friends to be going through something like that at the same time. I therefore needed three strangers who would be in a situation where friendships would be quickly forged. I’d attended a beach-based bootcamp for several years and intense friendships had formed speedily so I combined those two ideas.
The Hedgehog Hollow series was partly inspired by my auntie who runs a small scale hedgehog rescue operation and an assignment in my MA in Creative Writing where I needed to put a character in an uncomfortable setting. It was only going to be one book but it sprouted wings. Three have already been released and there are plans for two or three more plus a prequel!
💮Tell us about your writing day - are you disciplined, strictly 9 til 5, or are you more of a have a cup of coffee and think about it sort of writer?
Eek! No I’m not!
I used to be really disciplined. When I had my day job, my only possible writing time was evenings and weekends so I would knuckle down. Now that I’m writing full-time, it’s easy to get distracted. I spend long hours at my desk but I sometimes don’t spend them constructively so it’s something I’m working on.
I have a routine every morning where I see if I have any new reviews, check chart positions, and see what’s happening in the Top 100 on Kindle as it’s always interesting to see what’s moving at the top of the charts. And then I start work but get far too distracted by social media even though I’m not being particularly constructive with it. Far too often, I find I’m scrolling aimlessly!
It’s not unusual for me to be still at my desk working at 10.30pm and on weekends and I’m very aware of my lack of work:life balance so I’m wanting to get a better routine going where I do a 9 til 6 day and have my evenings free unless I’m at a deadline. Will I manage that? Mmmm. It’s difficult when writing is my job but it’s also the thing I love and would choose to have as my hobby!
💮Can you tell us if you have another novel planned?
Lots of them!
My final backlist book – Christmas Wishes at the Chocolate Shop – is out on 3rd August with a fresh edit and several new chapters. It’s the final book in my original 12-book contract with Boldwood Books.
I’ve just finished my second round of edits on book 13 – Snowflakes Over The Starfish Café. This is the start of a new 12-book deal and is set in a café just outside Whitsborough Bay. It’s out on 31st August and is the start of a trilogy although the story being told in the first book is completely wrapped up in that book.
There’s more to come from my Hedgehog Hollow series with book 4, A Wedding at Hedgehog Hollow – out on 6th January 2022 and the fifth book probably out in the summer.
And finally for fun! 😊
😊 Tell us four essential things every writer needs!
The serious answer: Imagination, resilience, patience, support
The fun answer: a comfy chair for the copious hours sitting at your desk, plenty of chocolate/cake/coffee (depending on what your fix is), a teddy bear to hug when you get negative reviews, and oodles of pretty stationery!
😊 If your life was a book, what would be its title?
Ha ha! What a brilliant question! I’ve had such an amazing year with my writing that I’d probably have to pick something along these lines:
The Girl Who Dared to Dream
When Wishes Come True
Maybe a bit cheesy but this past year has absolutely seen my dreams come true and I have to pinch myself every day to believe it!
😊 If you could be a character in one of your books who would you be, and why?
Such a tricky question! I tend to give them all amazing book boyfriends with whom I fall madly in love so they’re all very lucky, even if they’ve had a bit of a rough ride before that point!
If pushed, I’d probably be torn between Jemma running the teddy bear shop Bear With Me in All You Need Is Love and Samantha running Hedgehog Hollow. But I think Samantha might have the edge because of the setting and the work she does rescuing the hedgehogs. Every time I write a scene set on Thomas’s bench overlooking the wildflower meadow, I have such a sense of peace and tranquillity and long to be there in real life.
Jessica, where can we follow you on social media?
Jessica lives in Scarborough on the stunning North Yorkshire Coast in the UK. Her home inspired the creation of the fictional seaside town of Whitsborough Bay where she sets many of her books although the Hedgehog Hollow series takes readers to a gorgeous new countryside setting in the Yorkshire Wolds.
She lives with her husband, their teenage daughter and sprocker spaniel, Ella. She’s a stationery addict with a notepad obsession who loves chocolate (although it doesn't love her), hedgehogs, 80s music, collectible teddy bears and lighthouses.
Thank you so much for giving me the opportunity to join you as a guest on your fabulous blog, Jo. Really appreciate all the lovely support.
Thank YOU for being such a lovely guest.

domain: literature


By Peter Galuszka
Long-simmering immigration issues are starting to bubble over now that the U.S. Supreme Court has given a partial victory in opposing Arizona’s racist law. The ruling follows a bold action by President Barack Obama to allow law-abiding young people who happen to be undocumented aliens to stay in this country.
The court ruling rejected most of Arizona’s law except for allowing police to check the citizenship of anyone they arrest and suspect may be in the U.S. illegally. Obama’s action to allow some illegal aliens 30 years old and younger to remain in the U.S. is a humane and clever end run around the reactionary GOP-controlled House of Representatives.
Republican candidate Mitt Romney said he backs strict immigration enforcement and thinks states should be able to take matters into their own hands if the federal government fails to do so. Yet Romney has soft-pedaled immigration generally and is courting U.S. Sen. Marco Rubio, a Floridian born to Cuban immigrant parents as his possible running mate. If that happens, Romney is shorting Gov. Robert F. McDonnell to try to get the Latino vote.
The news shows just how far immigration has morphed as an issue. Virginia has been a battleground, notably with hard-right politician Corey Stewart’s anti- immigrant law in Prince William County which foreshadowed similar racist rules in Arizona and Alabama. Stewart, thankfully, is sputtering into history as the virulent anti-immigrant attitudes that colored the conservative movement two years ago are fading.
Also in Virginia, consider the story, front-paged in The Washington Post a few weeks ago that showcased the plight of young immigrants who arrived in this country at a very young age and have prospered since.
One is Heydi Mejia, an 18-year-old living in Chesterfield County who was brought to this country illegally when she was four years old. Mejia graduated with awards from Meadowbrook High School this month as a member of the National Honor Society. Her home had been raided a few months before by immigration officials who had arranged to have her deported to Guatemala a few days after she got her diploma. She got a year’s reprieve after the Post’s story. A few days later, Obama decreed that law-abiding illegals 30 years and younger who came to this country through no fault of their own may stay.
This is the kind of compassionate justice this country needs to heal the hatreds stoked by the anti-immigration types. The issue spans both parties. George W. Bush, for example, came up with a decent, long range immigration proposal that ended up being shot down. Later, his party started pandering to white reactionaries by provoking actions against undocumented workers that were in many ways imaginary. Company bosses in places like Arizona soon found they no longer could get workers, especially for the hard and dirty jobs.
In Virginia, one unfortunate view comes from George W. Grayson, a former delegate and William & Mary professor. Addressing Obama’s move he wrote in a Richmond newspaper that granting clemency to young illegals somehow hurts the “real” Americans who have trouble finding work. Conveniently, he forgets that young foreigners often do the jobs that “America’s” youth don’t want. For evidence, go to the Outer Banks this summer and listen to how many grocery cashiers and waitresses have accents from Russia, Poland or Ukraine.
One of Grayson’s more idiotic points is that we shouldn’t help Mexicans because they come from a resource-rich and corrupt nation. By that logic, the vast majority of America’s people should not be here.
The good news is that the mood on immigration is changing. Eventually we should get the kind of comprehensive reform that helps our economy and cements our position as a humane and just country.


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Our discounted price for culturing and DNA extraction is $600 for institutional billing and self-pay pricing. If cells fail to grow in culture, we will charge a reduced fee of $350 and will supply DNA from the direct tissue specimen to the performing laboratory, if appropriate/available.
We can provide shipping kits free of charge to providers within the contiguous United States. For US providers (excluding Alaska and Hawaii): To order a shipping kit, please provide the name, address, and phone number of the person we will be shipping to in an email to firstname.lastname@example.org. If the patient has an appointment set for the skin biopsy, please provide the date so we can ensure the kit arrives prior to the appointment. For providers in Alaska, Hawaii, or outside the US: Please contact us at at email@example.com or 844-255-3532 with any questions about optimal shipping conditions for specimens.
We extract from the skin biopsy then culture. Our priority is to attempt to obtain DNA from cultures. We will maintain cultures as a backup until testing is complete or until cultures are no longer healthy.
Each sample should come with an Allele Diagnostics requisition form Fibroblast Test Requisition Form requesting culturing and DNA extraction. In addition, each sample should include a completed test requisition for the laboratory that will be performing testing on the cultured DNA sample.
We will automatically inform a client if a culture fails. If you would like to be informed of successful culture extraction and shipping, please specify that on the requisition and include a reliable method of contact (phone number or email).
Allele offers institutional billing and self-pay. We do not bill insurance. If a patient opts for self-pay, we require payment information prior to processing the specimen. Credit card information may be provided with the specimen, or the patient may call the laboratory and provide a credit card number in advance of receiving the specimen. The discounted price for patient-pay and institutional billing is the same.
Skin biopsies are very fragile specimens. Tissue culture success can depend on numerous factors including age
of the specimen, age of the patient, shipping conditions, etc. To maximize the likelihood the specimen will grow
in culture, we recommend using an Allele-supplied kit or shipping label to ensure the sample arrives in the lab
the day after collection. We also ask for specimens to be collected under sterile conditions and shipped in
RPMI or sterile saline. The recommended size skin punch is 2x2 or 3x3 mm. If using your own label/kit, we
recommend overnight shipping, with Saturday delivery marked if sending late in the week. Please use the
following shipping address:
- Allele Diagnostics
- 120 N. Pine St., Ste. 152
- Spokane, WA 99202
Allele does NOT have a New York State License, so we cannot accept specimens from New York.

domain: medical


text:
X Factor: what history, hip hop and human interaction can teach us about evidence in public health policymaking
Sridhar Venkatapuram reflects on our unique ‘X Factor’ style event, which aimed to expand and diversify the evidence base for public health interventions and policymaking.
Over the past 5 years of my life, I have been a renter. My experience of living in rented accommodation has not often been great. Mould, damp, cramped conditions and absentee landlords aren’t unusu...
What the UK would look like if we were all eating more veg?
Our Director of the Healthy Lives Strategy asks: what if your life was always like winter?

domain: sociology


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Here are some of the more recent dramas you can view. It is only a couple of years old. That’s fairly younger in contrast to the more mature variations which is a lot much more mature. The new ones are just as fun as the previous types and perhaps even much better. It may be focused for a younger audience. Here are the ideas.
Look at it this way. Envision yourself becoming on your own and just minding your own business. All of a sudden, someone comes over and asks you for money. Now, compare this to a situation exactly where somebody arrives more than to you and Offers you cash. Now, which of the two options will you be likely to enjoy free download drama korea much more? Anyone who doesn’t select choice 2, drop me an e-mail and I’ll give you my bank account particulars.
The second woman. She is the girl you love. This is the initial sensation, when you initial noticed her heart began to throb, her shallow a smile you can really feel the globe is sunny flower leaves to come. You start blushing perspiring, so you understand that you have started a great journey.
Then 1 day, because some issues to you. Miles lengthy distance, or the university or of another person. You work difficult to sustain the relationship broken, but actually has struggled prior to breaking. When you are acquainted with her physique totally, sexual starvation reduction following you can’t discover any fresh, you don’t know what to say about her, don’t have any interest to take her to do some thing new. Two people suffocate in dull environment, fairly speechless. Split up is essential. Perhaps partnership damaged when you also will be unhappy sad, but the discomfort will not let you pain, that is relieved and loss of sensation. You feel pain, that is you should really feel the discomfort.
Maybe she promised, maybe noncommittally, so do you believe that might be agreed to, you determined grasp all the evidence to show her adore you, at the exact same time by another some negative hints of burnout. But it can’t block you, at this time, a coward also grew to become a hero, massive enthusiasm over shyness and dealing with a perfect embodiment of shame. So enthusiasm into motion unprepared, calm, sensible and intelligent all vanish, also misplaced self, all turned out to be a guide to action “will she like me so”?
All In: 1 of my all time preferred drama that you ought to watch. A lot of motion and love, which tends to make it extremely sad as well. It’s about a boy who was in adore with this girl. The boy goes to jail simply because of mischief and becomes a bodyguard in the near future. The girl on the other hand becomes a on line casino vendor and is great at it. They soon satisfy and fall in adore. View the adventure the two of them experienced to take to find every other.
Song #3: She Functions for the Money by Donna Summer. Remind yourself once more, why you bother operating so hard. This tune is so apt for any feminine employee’s Monday Early morning that they could function any woman in what ever clothing they gown for this music video clip. You work difficult for the money, so they much better treat you correct.

domain: psychology


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In the passage from gary soto’s book a summer life soto realizes this and shows his guilt by repeating the word “sin. Read carefully the following autobiographical narrative by gary soto then, in a well-written essay gleaming and the juice of guilt wetting my underarms. Throughout the autobiographical narrative written by gary soto, many different literary elements are used to recreate the experience of his guilty six-year old self. Advanced placement language & composition gary soto reveals his guilt through syntax,diction and imagery it is through his masterful use of technique. “the pie” summary gary soto tells the reader that it is a metaphor and talks about on top of that guilt gary didn’t share with crossed-eyed.
Gary soto post assessmentguilt is the price we pay volitionally for doing what we are going to do anyway -isabelle holland guilt is something we urinate for ourselves. This autobiographical narrative passage from a summer life by gary soto is a story of a six year old boy stealing pie and realizing the amount of guilt that his sin has caused him to feel. The never-ending guilt of stealing many times in life, people do things that they regret doing later on, and it often comes back to haunt them. Idle time is the devils time as the narrator, gary soto recreates a childhood experience in which he steals a pie from the german market although stealing a single pie might seem.
List five words, phrases or images which evoke a sense of guilt why is gary soto feeling guilty the last line of paragraph 4 – “a burp perfumed the air. Good purpose paragraph with movement gary soto’s purpose in writing this narrative is to describe the effects that sin has on a person he starts out by describing the initial guilt that. Commentary on an excerpt from a summer life young gary soto recreated a childhood experience in his soto expresses his tone of remorse and guilt. Author, gary soto within a young self-proclaimed innocent boy ultimately, the contrast, imagery, and repetition serve to express soto’s guilt.
View gary soto's narrative from english la ap english at coral gables senior high school de la rosa 1 de la rosa, natalie ap english per 01 gary soto’s guilt. Thinh au sotos a summer life rhetorical analysis everyone is familiar with guilt gary soto is not an exception. 75 readings plus10 th edition gary soto: like mexicans chapter 7 example and illustration white guilt barbara dafoe whitehead. Honors seniors: prose analyses please submit remembering his childhood guilt, gary soto uses perceptible imagery and relatable symbolism to describe.
Gary soto guilt essay, рефераты - иностранный язык - gary soto analysis essay research paper the eve plucked that apple and gary soto stole that pie thinking only of the immediate. Guilt and remorse are two main feelings that people may understand differently, whether on account of past experiences, learning tactics, or an opinion on religion in the narrative a summer.
This lesson focuses on teaching students to annotate a text when students annotate a text, it helps them to comprehend and analyze what they are reading this packet contains two poems, the. In this autobiographical narrative, gary soto recreates an experience of his guilty six year old self the purpose of gary soto s narrative is to explain. The no guitar blues author: gary soto summary: the owners are grateful and press a twenty dollar bill into his hand seized by guilt over his deception.
Gary soto, in his passage from a summer life, depicts the guilt of his six-year-old self during a pie-stealing escapade. Gary’s soto’s the pie gary soto manages to bring his guilt to life he reveals a judgment on guilt and forgiveness, on a quickly spreading ignorance of guilt. List five words, phrases or images which evoke a sense of guilt b why is gary soto feeling guilty 4 the last line of paragraph 4 – ―a burp perfumed the air. Analysis essay soto: 1996 in the passage from “1996”, gary soto’s continuous thoughts of guilt convey themselves through a shameful tone. Gary soto's the pie literary analysis on gary sotos the pie prominent soto compares the sweat from his restiveness to the juice of guilt. Start studying pie story learn gary soto main point to show but he is enticed by the pie and after he eats it he feels guilty and knows that there is no.
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domain: artistic


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Item Availability & Lead Time
While many of the items in this catalog section of our website are on display in our store and in stock, many are available by special order. Please contact us for availability and pricing information .
Special Order Lead Time:
View recently taken pictures of nearly all the items on display in our store.
Casual Dining Room Group Item Description
The Tabitha Casual Dining Room Group by Coaster is available in the area from FMG - Local Home Furnishing.
Tabitha Collection Description
Turn back time with the glorious design of the Tabitha dining collection. Rich birch solids and veneers are set in a Cherry finish. A traditional style comes alive with intricate carvings, sculpted bases and legs, comfortable upholstered seat cushions and brushed metal hardware. Find the right look for your home with the dining arm and side chairs, rectangular dining table, round glass top dining table, buffet, hutch and complete china cabinet.
The Tabitha collection is a great option if you are looking for Traditional furniture in the area.

domain: design


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New NSW Fair Trading disclosure obligations will start to bite
Suppliers can expect NSW Fair Trading to more actively monitor and enforce new disclosure obligations under the Fair Trading Act 1987 (NSW). These obligations require suppliers to take extra steps to disclose certain terms and conditions to consumers.
The new requirements were introduced in July 2020 and, after a grace period during which NSW Fair Trading took an education and awareness raising approach to compliance, we expect the state regulator will be more closely scrutinising businesses, particularly their online terms, from now on.
The new provisions require businesses to “take all reasonable steps” to ensure consumers are aware of the “substance and effect” of terms and conditions which “may substantially prejudice” their interests.
For more details about what this means for you (as a consumer) and/or your business if you are in or supply to consumers in NSW, read our client alert here.
Pecuniary penalties and other enforcement action can apply if a supplier does not provide adequate disclosure of a prejudicial term, with NSW Fair Trading having power to take action.
We are helping clients understand what these obligations require of them, in light of the particular nature of their businesses and the ways sales are made to customers.
If you supply consumers directly, particularly online, we recommend reviewing all your terms and conditions in light of these new obligations. We are undertaking these reviews for clients and would be pleased to help.
Subscribe to stay up to date with the latest developments

domain: law


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Director: Sülbiye V. Günar
Feature film | D | 2002
94 min | Dolby SR
36th Hof International Film Festival
Johanna calls herself Jeanne and is fed up with school. She’d much rather study fashion in Paris. The only problem are the horrendous school fees. Not only is her mother dead against her daughter going to Paris, she’d also never be able to raise the money.
When, belatedly, Johanna asks for maintenance from her father, she finds out he is not her real father at all: her mother confesses that her natural father is a Turk, Cumhur, who has a family and runs a posh restaurant in Cologne. Financial pressure makes Johanna apply as kitchen help at her father’s place, not revealing her relationship to him. Very soon, she’s part of the Turkish family, especially since she knows Cumhur’s daughter, Idil, from school.
She feels quite happy with her new family, but should she reveal the truth, she’s certain there’d be no place for her. So she sticks to her original plan to steal the money for going to Paris from her father. When one night she puts plan to action, she is intercepted by Cumhur who meanwhile has been made wise by Johanna’s mother …

domain: entertainment


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The apartment is located in Cavtat, a quiet little town with rich cultural and historical heritage, beautiful beaches and scenery, just 20 minutes ride to Dubrovnik's Old Town.
Two-bedroom apartment features free WiFi, air-conditioning and SAT TV, fully equipped kitchen with dining area, living room with sofa bed and two private bathrooms. It will also provide You with a lovely private terrace and sea view.
The kitchen includes refrigerator, oven, stove top, toaster, freezer, electric kettle and kitchenware.
Free parking is available on site. Shared washing machine will be at guests' disposal.
This location is perfect for guests who love to enjoy the sun and the sea, being close to Dubrovnik, but far enough to avoid the crowds and keep their peace and privacy. Apartment is located 20 minutes walk to the nearest beach. Various restaurants, bars and shops can be found on the famous seaside promenade, ideal for those who enjoy the nature and long sunset walks.
There are few historical places which you should visit while staying in beautiful Cavtat. One of the places is the Franciscan Monastery built in the 15th century, as well as the Monastery of family Racic built in the 20th century on the initiation of the widow Racic after many tragic events, and she said ''My heir will build on the church graveyard in Cavtat grave with a chapel, in whom will rest my husband, our children and me. " There is also a famous Art Gallery Bukovac and the famous statue of Baltazar Bogisic.
Old Town of Cavtat is situated at the south slopes of the peninsula. Known as Epidaurus in ancient times, today it is the tourist and cultural center of Konavle, the southernmost region of Croatia. In the times of the Dubrovnik Republic, Cavtat was the second most important seaport, place of trade and of administration in the Republic after Dubrovnik.
Old Town of Dubrovnik is 17 kilometers away.
Dubrovnik Airport is 4 km away.

domain: geography


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Women's lightweight boxer, Christy Martin, of Orlando, Fla., displays her World Women's Boxing Association lightweight championship belt and a statue of herself her aunt made as she stands outside her home Sunday, Oct. 22, l995. Martin, who was a school teacher after graduating with a BS in education from Concord College in Athens, W.V., says she got into boxing to fill the void of college and being a basketball player. "I love the excitement of the crowd," says Martin. I like being the positive figurerather than the villian."
LENNOX MCLENDON, ASSOCIATED PRESS
Copyright © 2018, Los Angeles Times

domain: news


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SadMan Software: Sudoku is a tool to help you solve puzzles.
Okoker software, Inc.
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Game Zone Project
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Sudoku Master is the best Sudoku game for the PC.
Mega Sudoku Plus provides unlimited Sudoku puzzles to solve with levels...
Sudoku Works is a simple but very comprehensive sudoku game.

domain: hobby


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In the beginning of October Kensington’s Asia Regional Manager Michael Bertuzzi took some well-deserved time off to return to China (where he lived from 2004 - 2007 running an outdoor adventure company that, like Kensington Tours is listed by National Geographic as top 100 in the world) to get married. Michael came up with a stunning itinerary for the 20 something family and friends that will be celebrating in Yangshuo with him. A fusion between our 10 day Family Adventure and 16 day Signature China tours, this customized programme allows guests to explore the highlights of this ancient civilization.
Wedding party and guests from North America will experience the diversity of China starting with the culture of Beijing where they’ll enjoy the stunning Hilton Wangfujing, ranked #1 out of 2726 hotels.
After a couple days taking in the culture and history of Beijing they are off to the rural splendour of picturesque Yangshuo in the Guilin area (an idyllic wedding backdrop) where they will stay at the intimate Li River Retreat.
This family run hotel isn’t 5-star but its scenic location and caring ownership is unmatched and always a favourite. Surrounding a day of matrimonial celebrations, guests will enjoy 4 days of tours, picnics, bicycling, boating, climbing, trekking and more, including the Liu San Jie evening show created by Oscar winner Zhang Yimou and learning how to cook local cuisine at a cooking school. After this, the majority of the guests are headed off on custom trip extensions including Michael’s favourite; the 3 day Dragons Backbone hike through the Longsheng Rice Terraces. All travelers will depart from the modern metropolis of Shanghai where they will be staying at the magnificent Le Royal Meridien.
Stay tuned for a China update soon after Michael returns! Look forward to great new travel ideas, trends, properties, private guided tour ideas and more for 2010.
This exciting six day discovery includes the colonial cities of Leon and Granada, colorful markets, scenic lakes and volcanoes.
(Tokyo, Hakone, Kyoto)
Embark on this exploration of Japan's highlights, with the support of local guides, comfortable train transportation, and enough down time to truly get lost in the land of the rising sun.
(Mountain Pine Ridge)
Bask on sandy white beaches, snorkel with sharks and stingrays, and enjoy the relaxed pace of life on Ambergris Caye. Take the relaxation up a notch, when you board your private catamaran Aubisque, for a 3 night sail around the Cayes.
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Officials from the European Union and India agreed Friday to take decisive and concerted action against global terror organizations, including Islamic State (IS) and al-Qaeda.
The two sides also vowed to fight pro-Pakistan organizations Lashkar-e-Taiba (LeT), Jaish-e-Muhammad (JeM) and Hizbul Mujahideen (HM) who claim to be fighting for Muslim-majority Kashmir’s freedom from Indian rule. An outbreak of a separatist insurgency in the disputed Himalayan region, claimed in its entirety by both India and Pakistan, has claimed more than 70,000 lives since the late 1980s.
A joint statement issued at the end of the 14th EU-India Summit condemned recent terrorist attacks in Indian Kashmir, other parts of India and Europe, with both sides promising to bring the perpetrators to justice. Prime Minister Narendra Modi hosted the meeting in New Delhi attended by Donald Tusk, president of the European Council, and Jean-Claude Juncker, president of the European Commission.
The two sides resolved to step up cooperation to prevent and counter terrorism, violent extremism, radicalization, recruitment and to dismantle terror infrastructure and funding, according to the statement.
“The leaders agreed to strengthen cooperation to take decisive and concerted actions against globally proscribed terrorists and terror entities …,” the joint statement said.
The list of proscribed militants included Hafiz Saeed, a Pakistan-based cleric and founder of Lashkar-e-Taiba, which Indian authorities have blamed for the November 2008 terrorist attacks that killed more than 170 people over four days in Mumbai. Dawood Ibrahim, a fugitive who is wanted by India for the country’s deadliest terrorist attack, the 1993 Mumbai bombings, was also on the list.
And in a reference to the Rohingya community, India and the E.U. urged Myanmar authorities to implement the Kofi Annan-led Rakhine Advisory Commission’s recommendations, and work with Bangladesh to enable the return of displaced persons from all communities to Rakhine state.
“The world responding to Myanmar to change its course and become tolerant toward the Rohingya community is a good development,” Bharat Karnad, a foreign policy analyst with the New Delhi-based Center for Policy Research, told BenarNews.
But other analysts said India was playing the diplomatic card.
“India’s position on Rohingya is diabolic. It is asking Myanmar to take them back but is not condemning its army for the human rights violations it is committing against the minority,” said Sai Balaji, a researcher in the Diplomacy and Disarmament Division of the Center for International Politics at New Delhi’s Jawaharlal Nehru University.
In August, India’s ruling Hindu-nationalist Bharatiya Janata Party said it planned to deport about 40,000 Rohingya refugees settled in the country – a move slammed by human rights organizations worldwide.
During a hearing in the Supreme Court last week, the government said it would submit evidence that Rohingya living in India had links with terror organizations.
Dushyant Nagar, a New Delhi-based political affairs analyst, said the Rohingya issue was a sensitive matter for India.
“India has to balance between its constitution and the human rights while dealing with Rohingya,” he told BenarNews. “The government should cautiously study and verify intelligence whether the Rohingya are actually connected with terror groups. If they are involved, they should be pushed out and if they are not, they need to be dealt with in a humane manner.”


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Last month I attended the trial in Staines magistrates’ court, of a group of activists charged with offenses relating to the shutting down of five Shell petrol stations back in July. The activists were from Greenpeace, and their actions against the oil giant Shell were part of a day of action which saw 78 Shell stations shut down in London and Edinburgh, and part of a week of action against Shell with direct action and protest taking place in over 30 countries, involving thousands of people. The people in the dock in Staines that day ranged in age from their early twenties to late fifties, among them a care worker, a foster mother to four children, and a playwright.
The week of action was and is part of a sustained campaign against oil drilling in the fragile and pristine Arctic, home to unique species like polar bears and narwhals, to indigenous communities who live in ways unchanged for thousands of years, one of the last wild places on earth. The Arctic campaign encompasses, not only direct action against Shell and other oil companies, but also the gathering of over 2 million signatures of people who want to declare the Arctic as a global sanctuary; relationship building with Arctic communities; the enabling of scientific research on Greenpeace ships; and a political process – through the Arctic council and the UN – to try and reach global agreement that the area around the North Pole must be off-limits to industrial exploitation.
Sometimes when we talk about peak oil, we put aside the fact that there is still great deal of oil in the ground and under the sea, enough left to fry the planet several times over, and much of it difficult and dangerous to extract – deep in oceans, under the fast-melting Arctic ice, absorbed into Canadian tar sands. The technology to reach this oil is improving and the economic incentives to do so are becoming greater. So as we Transition on one hand to a society that uses less oil, on the other we urgently need to stop the companies from extracting every last barrel of oil on the earth.


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With almost 30 years of experience in the industry, IHC Hytech specializes in the design and manufacture of high quality, professional diving and hyperbaric equipment such as hyperbaric chambers and decompression chambers. We produce a wide range of products and each one is designed to meet the highest standards. Every product is supported by extensive quality control and after-sales service for maintenance advice and spare parts. We are the partner of choice for innovative, sustainable and integrated diving, and life support equipment. With our extensive knowledge and in-house design capabilities, we ensure compliance with the latest technological developments, strictest safety regulations and most stringent environmental standards.read more
Maintaining a healthy atmosphere inside a hyperbaric chamber
The innovative IHC Hytech CO2 scrubber has been specially designed to remove CO2 from the atmosphere of hyperbaric chambers. The scrubber is equipped with an induced flow fan which pulls the polluted air through a canister filled with soda lime to adsorb the CO2.
Hyperbaric chambers are sealed pressure vessels. This means that the atmosphere inside the hyperbaric chamber quickly becomes polluted with CO2 from exhaled air. High concentrations of CO2 can be poisonous and must be removed from the chamber atmosphere.Read full article
Underwater communication is critical for safe diving operations. The tender must be able to communicate with the diver(s) at all times and vice versa. If the weather conditions or circumstances on the surface suddenly change, the tender will need to communicate with the diver to abort the diving operation.Read full article
The IHC Hytech line of reducers has been specially engineered for applications that require light-weight, corrosion resistant, compact and reliable reducers in high pressure systems. For optimal performance, the reducers are made to customer specifications. The pressure can be set between 7.5 and 30 bar.Read full article

domain: chemistry


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Two timelapse films for you today to finish off the fascinating trip we made from Windmill End on the Dudley No. 1 Canal to Birmingham. The first one is only nine seconds long - I don't know why - but does include Telford's Galton Bridge and the Galton Tunnel.
The last part of day nine of our April 2011 cruise is recorded in the following 1'08" timelapse film which takes us under the amazing Engine Arm Aqueduct and on to central Birmingham, just past Gas Street Basin and Worcester Bar. We tied up in front of The Mailbox, just before the right turn to Holliday Wharf.
A Walk To Grindley Brook - Our overnight mooring We left our overnight mooring before Bridge 38, Eric steered the boat while Ben and I walked. We had some gun shots last night I think...
2 hours ago


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Sarah Michelle Gellar Shares Epically Retro Throwback Pic With Kelly Ripa
Big hair, don't care.
Sarah Michelle Gellar poked fun at her style in a throwback photo of herself with Kelly Ripa, which she shared on Instagram on Thursday. In the pic, a brunette Buffy with bangs smiles wide while wearing a white halter top dress and heavy makeup. Ripa, meanwhile, exudes beauty queen vibes in a sequin turquoise strapless gown with her blonde locks half up in a big poof.
"They say big hair, big dreams....maybe they were right. Big hair and clearly didn’t care - ha maybe it finally helped me hit 5’5” ( ?) @kellyripa #throwbackthursday," Gellar, 41, captioned the throwback Thursday shot.
Ripa jokingly replied to a comment from writer Gary Janetti, writing, "That was from Duchess Fergie's wedding I believe. I know because I wore fake hair and a crooked smile."
Gellar and Ripa launched their careers on the soap opera All My Children back in the early '90s, and have remained close friends ever since. The Live With Kelly and Ryan co-host also met her husband, Mark Consuelos, on the same daytime soap in 1995.
Watch the video below for another epic throwback to Ripa and Consuelos' 1996 wedding.
Freddie Prinze Jr. Helps Sarah Michelle Gellar Celebrate Instagram Milestone With a Cake Smash
Sarah Michelle Gellar Jokingly Says She Bit Beyonce With Funny ‘Buffy’ Throwback Pics!
Kelly Ripa Reveals She and Husband Mark Consuelos Briefly Broke Up a Week Before Their Vegas Wedding

domain: celebrity


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Emerald Expositions Events (EEX) Receiving Somewhat Positive Media Coverage, Analysis Finds
News stories about Emerald Expositions Events (NYSE:EEX) have trended somewhat positive this week, Accern Sentiment Analysis reports. The research group ranks the sentiment of news coverage by analyzing more than 20 million news and blog sources. Accern ranks coverage of companies on a scale of -1 to 1, with scores nearest to one being the most favorable. Emerald Expositions Events earned a media sentiment score of 0.17 on Accern’s scale. Accern also assigned press coverage about the company an impact score of 46.2188825057384 out of 100, meaning that recent news coverage is somewhat unlikely to have an effect on the stock’s share price in the next several days.
Separately, Zacks Investment Research raised shares of Emerald Expositions Events from a “sell” rating to a “hold” rating in a report on Thursday, September 28th. Four analysts have rated the stock with a hold rating and five have given a buy rating to the company’s stock. The stock currently has an average rating of “Buy” and a consensus price target of $24.28.
Shares of Emerald Expositions Events (NYSE EEX) traded down 0.47% during mid-day trading on Thursday, reaching $23.21. 23,668 shares of the company’s stock were exchanged. The stock has a market cap of $1.68 billion and a price-to-earnings ratio of 89.96. Emerald Expositions Events has a 52-week low of $18.67 and a 52-week high of $24.56. The stock’s 50-day moving average is $22.31 and its 200-day moving average is $21.85.
Emerald Expositions Events (NYSE:EEX) last released its quarterly earnings data on Thursday, August 3rd. The company reported $0.18 EPS for the quarter, hitting the Zacks’ consensus estimate of $0.18. The company had revenue of $74.14 million for the quarter, compared to the consensus estimate of $74.81 million. On average, equities analysts expect that Emerald Expositions Events will post $1.06 EPS for the current year.
About Emerald Expositions Events
Emerald Expositions Events, Inc (Emerald), formerly Expo Event Holdco, Inc, is an operator of business-to-business (B2B) trade shows. The Company operates across multiple sectors, which includes: Gift, Home and General Merchandise; Sports; Design and Construction; Technology; Jewelry; Other Trade Shows; and Other Events.
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When BSE, or mad cow disease, surfaced in England in 1986, Americans learned that cattle, which are herbivores, were routinely being fed the flesh of other cattle; the practice helped keep meat cheap but at the risk of a hideous brain-wasting disease.The 1993 deaths of four children in Washington State who had eaten hamburgers from Jack in the Box were traced to meat contaminated with 0157: H7, a mutant strain of the common intestinal bacteria first identified in feedlot cattle in 1982.But there are indications that these various voices may be coming together in something that looks more and more like a coherent movement.Many in the animal welfare movement, from PETA to Peter Singer, have come to see that a smaller-scale, more humane animal agriculture is a goal worth fighting for, and surely more attainable than the abolition of meat eating.Most importantly, the price of food came down, or at least the price of the kinds of foods that could be made from corn and soy: processed foods and sweetened beverages and feedlot meat.(Prices for fresh produce have increased since the 1980s.) Washington had succeeded in eliminating food as a political issue—an objective dear to most governments at least since the time of the French Revolution., criticize supporters of “sustainable” agriculture—i.e., producing food in ways that do not harm the environment—for advocating reforms that threaten to raise the cost of food to the poor.Animal rights advocates occasionally pick fights with sustainable meat producers (such as Joel Salatin), as Jonathan Safran Foer does in his recent vegetarian polemic, Eating Animals.
Beginning in the late 1980s, a series of food safety scandals opened people’s eyes to the way their food was being produced, each one drawing the curtain back a little further on a food system that had changed beyond recognition.
The administration’s cheap food policy worked almost too well: crop prices fell, forcing farmers to produce still more simply to break even.
This led to a deep depression in the farm belt in the 1980s followed by a brutal wave of consolidation.
Joel Salatin, the Virginia meat producer and writer who has become a hero to the food movement, fulminates against food safety regulation on libertarian grounds in his Everything I Want to Do Is Illegal: War Stories From the Local Food Front.
Hunger activists like Joel Berg, in All You Can Eat: How Hungry Is America?
Cheap food has become an indispensable pillar of the modern economy.


42 U.S. Code § 292w - Procedures for appeal of termination of agreements
In any case in which the Secretary intends to terminate an agreement with a school under this subpart, the Secretary shall provide the school with a written notice specifying such intention and stating that the school may request a formal hearing with respect to such termination. If the school requests such a hearing within 30 days after the receipt of such notice, the Secretary shall provide such school with a hearing conducted by an administrative law judge.
Source(July 1, 1944, ch. 373, title VII, § 727, as added Pub. L. 102–408, title I, § 102,Oct. 13, 1992, 106 Stat. 2020.)
A prior section 727 of act July 1, 1944, was classified to section 294 of this title prior to the general revision of this subchapter by Pub. L. 102–408.
Another prior section 727 of act July 1, 1944, was classified to section 293g of this title prior to renumbering by Pub. L. 94–484.


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D Game - Quiet PC You can use the Contents menu on the left to skip directly to the section you're looking for. D games in. 3D Game List and How to enjoy 3D game as below. Medieval 2 - Total War. You can check nVidia 3D Stereo Driver User Manual in below.
Medieval II Total War - SteamDB Alternatively, click the Enter button to access the manual, and use the Next and Previous buttons to navate page by page. There's even more information in the FAQs and Read Me: you can view them using the buttons at the top of the screen. App Type, Game. Name, Medieval II Total War. Store Name, Medieval II Total War. Developer, The Creative Assembly. Publisher, SEGA. Supported Systems.
Napoleon Total War Game Manual Battles - Scribd Immediately stop playing and consult a doctor if you experience any of these symptoms. NAPOLEON TOTAL WAR GAME ACTIVATION KEYCODE The Elite Regiment pack. The Elite Regiment pack includes 5 premium units 5 R i i 5e Regiment d.
Medieval II Total War Official Strategy Guide A very small percentage of people may experience a seizure when exposed to certain visual images, including flashing lhts or patterns that may appear in video games. BradyGames' Medieval II Total War Official Strategy Guide includes the. I've been gaming and purchasing gaming manuals since the mid-to-early 90's.
Steam Community Medieval II Total War Even people who have no history of seizures or epilepsy may have an undiagnosed condition that can cause these “photosensitive epileptic seizures” while watching video games. I just started the game tutorial today and in the beginning of the campain instruction it. 655. Medieval 2 Total War -- Street Fhting Man. These are the orinal manuals that came with the disk versions of the main game and the expansion.
Empire and Napoleon Total War Collection - Game of the Year PC. These seizures may have a variety of symptoms, including lhtheadedness, altered vision, eye or face twitching, jerking or shaking of arms or legs, disorientation, confusion, or momentary loss of awareness. Buy Empire and Napoleon Total War Collection - Game of the Year PC. Medieval 2 Total War Gold PC DVD by Games Outlet Windows 2000 / XP £. In reality, the manual should have been beefy, espcially as its supplied as PDF on the.
Creative Assembly A Beginners Guide to Medieval Seizures may also cause loss of consciousness or convulsions that can lead to injury from falling down or striking nearby objects. Thanks to Tricky Lady for compiling this PDF. 2campan game 2.1 Your main aims 2.2 Buildings and the tech tree. This guide has been written with Medieval Total War in mind. I will not be reiterating the manual in this guide, so go and read the manual cover to cover and then come back.
Deus lo Vult! mod for Medieval II Total War Deus lo Vult! mod for Medieval II Total War Kingdoms. and collecting governmental\military points to achieve an advancement during his game. Deus lo Vult 6.2 - Handbook/Manual in English language. PDF format.
Medieval 2 total war game manual pdf:
Rating: 100 / 100
Overall: 97 Rates


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Performance-enhancing DrugsBook - 2015
Pulling from a wide range of highly respected sources and publications, this collection of essays tackles the topic of performance-enhancing drugs. Readers take a look at whether or not the use of performance-enhancing drugs should be accepted, how dangerous these drugs can be, and the effectiveness of drug testing. This volume also discusses the future of performance-enhancing drugs.
Publisher: Farmington Hills, Michigan :, Greenhaven Press,,
Branch Call Number: 362.29088 P438o
Characteristics: 206 pages : illustrations ; 24 cm
Alternative Title: Performance enhancing drugs : opposing viewpoints


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Foot-and-mouth disease virus (FMDV) causes a highly contagious disease in cloven-hoofed animals. A synthetic vaccine candidate consisting of dendrimeric peptides harbouring two copies of a B-epitope [VP1(136–154)] linked to a T-cell epitope [3A(21–35)] of FMDV confers protection to type O FMDV challenge in pigs. Herein we show in cattle that novel dendrimeric peptides bearing a T-cell epitope [VP1(21–40] and two or four copies of a B-cell epitope [VP1(135–160)] from type O1 Campos FMDV (termed B2T and B4T, respectively) elicited FMDV specific immune responses to similar levels to a commercial vaccine. Animals were challenged with FMDV and 100% of vaccinated cattle with B2T or B4T were protected to podal generalization. Moreover, bovines immunized with B4T were completely protected (with no clinical signs) against FMDV challenge after three vaccine doses, which was associated with titers of viral neutralizing antibodies in serum higher than those of B2T group (p< 0.05) and levels of opsonic antibodies similar to those of animals immunized with one dose of FMDV commercial vaccine. Bovines vaccinated with both dendrimeric peptides presented high levels of IgG1 anti FMDV in sera and in mucosa. When IgA in nasal secretions was measured, 20% or 40% of the animals in B2T or B4T groups respectively, showed anti-FMDV IgA titers. In addition, B2T and B4T peptides evoked similar consistent T cell responses, being recognized in vitro by lymphocytes from most of the immunized cattle in the proliferation assay, and from all animals in the IFN-γ production assay. Taken together, these results support the potential of dendrimers B2T or B4T in cattle as a highly valuable, cost-effective FMDV candidate vaccine with DIVA potential.
Citation: Soria I, Quattrocchi V, Langellotti C, Gammella M, Digiacomo S, Garcia de la Torre B, et al. (2017) Dendrimeric peptides can confer protection against foot-and-mouth disease virus in cattle. PLoS ONE 12(9): e0185184. https://doi.org/10.1371/journal.pone.0185184
Editor: Paulo Lee Ho, Instituto Butantan, BRAZIL
Received: May 2, 2017; Accepted: September 7, 2017; Published: September 26, 2017
Copyright: © 2017 Soria 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.
Data Availability: All relevant data are within the paper.
Funding: Work at INTA was supported by the National Institute of Agricultural Technology (grant number PNSA 1115052) and cooperation agreement INTA-INIA (Argentina-Spain SA 20938). Evaluation of vaccine immunogenicity and protection in cattle. Work at Universitat Pompeu Fabra and CBMSO was supported by MINECO, Spain (grant number AGL2014-52395-C2). Dendrimeric peptides synthesis and article submission. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Foot-and-Mouth disease virus (FMDV) is a Picornavirus belonging to the genus Aphthovirus. The viral particle consists of a positive-strand RNA genome encoding four capsid proteins, VP1, VP2, VP3 and VP4, and eleven different mature non-structural proteins, NSP-3A among them . FMDV has seven immunologically distinct serotypes, namely O, A, C, SAT1, SAT2, SAT3 and Asia 1. This virus produces a highly transmissible and devastating disease of farm animals, foot-and-mouth Disease (FMD), which is the most feared animal disease worldwide, causing large economic losses during an outbreak.
There is great disparity in progress towards FMD control and eradication. While some countries are either FMD free or well on the road to achieving eradication, others are at an early stage of FMD control. In endemic areas, such as Asia Africa and South America, FMD control is performed by regular vaccination based on inactivated whole-virus. . The limitations shown by these conventional vaccines have prompted the study of new, safer alternative vaccines . Synthetic peptides are one of the most promising vaccine candidates for infectious disease, such as FMD, as they are highly pure, defined, stable and safe. Moreover, recent studies have shown that synthetic peptides achieve protective immunity against challenge in swine [3, 4]. On the other hand, differentiation between vaccinated from infected animals is feasible with such vaccines.
Linear peptides containing an immunodominant B-cell site located in the GH loop around at positions 140–160 of capsid protein VP1 [5, 6] can protect against FMDV challenge. However, the protection conferred by these peptides is limited due, among other factors, to the lack of T-cell epitopes [5, 7]. Therefore, an effective peptide vaccine needs multivalency of B-cell epitopes to elicit a high neutralizing antibody response and T-cell epitopes that provide adequate cooperation . Multiple antigenic peptides (MAPs), also named dendrimers, are radial or wedge-like branched macromolecules that contain a peptide core attached to a define number of epitopes [9, 10]. Pigs vaccinated with a dendrimeric peptide, which included the T-cell epitope 21–35 from 3A and four copies of the B-cell site VP1 [136–154], were protected against type C FMDV challenge . Recent studies indicated that a similar construct incorporating the B cell site sequence from type O FMDV O-UKG induced full protection in pigs .
In this report, novel dendrimeric peptides were designed containing a bovine T-cell epitope 21–40 from VP1  linked to two (B2T) or four (B4T) copies of the B-cell site VP1 [135–160] from FMDV O1 Campos assembled using a maleimide conjugation. A pilot study was performed to address the immunogenicity of such dendrimeric vaccines and the protection afforded in cattle against viral challenge.
Our results show for the first time that not only tetravalent presentation of B-cell epitopes linked to the T-bovine epitope but also bivalent formulation results in an effective vaccine that conferred protection in cattle. Thus, these synthetic peptides can be considered promising vaccine candidates with reasonable prospects of clinical application.
Materials and methods
Two peptides (Fig 1) incorporating a T-cell epitope [VP1 (21–40)] immunodominant for bovine lymphocytes [11, 12] and two or four copies of the B-cell epitope [site A–VP1 (135–160)], termed B2T or B4T, respectively, in a dendrimeric arrangement were synthesized as described [4, 13]. The C-terminal Cys side chain thiol is linked to Lys via a 3-maleimidopropionic acid unit; the peptides were purified by HPLC and characterized by mass spectrometry. Sequences are derived from FMDV O1 Campos.
FMDV O1 Campos was kindly provided by Biogenesis Bagó S.A. as binary ethylene-imine (BEI)-inactivated (iFMDV). Purified virus was obtained by a sucrose density gradient centrifugation method  and was used for ELISA and lymphoproliferation assay. For challenging and virus neutralization assays, infective virus (kindly donated by the Argentine National Service of Animal Health) was used in BSL-4 OIE laboratories and boxes at INTA.
Animals, vaccines, immunization and infection of cattle
Eighteen Hereford calves serologically negative for FMDV, approximately 6 months old, were used in the experiment. Groups of five animals were inoculated twice (day 0 and 18) by subcutaneous injection in the front left quarter with 2 mg of B2T or B4T peptide in 2 ml of a water-in-oil single emulsion. The adjuvant included was MONTANIDE ISA 50 V (Seppic). At 38 dpv animals were boosted with 0.5 mg of B2T or B4T peptide. Forty-six days after the first vaccination, immunized animals as well as 3 control unvaccinated bovines were challenged by nasal instillation with 1 ml (0.5 ml for each nostril) of a 104 suckling mouse lethal doses 50% equivalent to 104 Bovine Infective Doses 50% (BID50) of infective FMDV O1 Campos (determined by titration on cattle tongue) [15–17]. This method mimics a natural FMDV infection . Control unvaccinated bovine (n = 3) were challenged at the same time and following the same procedure. Seven days post challenge (dpc) all animals were checked for FMDV-induced lesions on feet, mouth and tongue. Animals with absence of FMDV lesions at the feet were considered protected against podal generalization (PPG). Another five calves were immunized by subcutaneous injection with a single dose of commercial FMDV vaccine, a water in oil single emulsion containing FMDV strains A Arg 2000, A Arg 2001; A24 Cruzeiro and O1 Campos. This vaccine had been approved by the Argentine Animal Health Service (SENASA) with more than 80% of expected percentage of protection against each of the viruses included in its formulation . This group was used as a control of immune response quality. Experiments were carried out according to INTA Ethics Manual “Guide for the use and care of experimental animals”. The protocol was approved by the Institutional Animal care and use Committee “Comité Institucional para el cuidado y uso de animales de experimentación” CICUAE INTA CICVyA (Permit Number: 77/2013).
Measurement of anti FMDV antibodies
An indirect ELISA was used for anti-dendrimer antibodies measurement. Maxisorp 96-well plates (Nunc) were coated with B4T peptide (30μg/ml), plates were washed and blocked with PBST-OVA 1% and dilutions of serum samples were added. After incubation, plates were washed and horseradish peroxidase (HRP) labeled goat anti-bovine IgGs antibody (KPL, USA) was added. After washing, ortho-phenylenediamine (OPD)-H2O2 was added as HRP substrate. FMDV-specific antibodies were detected by means of an indirect ELISA, as described . Briefly, Immulon II 96-well ELISA plates were coated with 2.6 μg/ml FMDV O1Campos and processed as described above.
The antiviral ELISA detailed above was modified in order to detect FMDV-specific IgG1, IgG2 (in sera), and IgG1, IgA (in nasal swabs) antibodies [20, 21]. After incubation with samples, a mouse anti-bovineIgG1, IgG2 or IgA monoclonal antibody was added (kindly provided by Dr. S. Srikumaran, University of Nebraska, USA). Lastly, a (HRP) labeled goat anti-mouse IgG antibody was added after wash. OPD was used as HRP substrate. Absorbance was recorded at 492nm (A492) in a microplate photometer (Multiskan FC, Thermo). The cut-off was established as the mean A492 of the negative sera (from 5 unvaccinated animals) plus two standard deviations (SD). Antibody titers were calculated as log 10 of last reciprocal dilution above cut-off. Positive control sera were included in every plate.
Inactivated FMDV (iFMDV) was labeled with FITC (Sigma, St. Louis,MO) as described . Briefly, FITC-iFMDV was incubated with serum and added to bovine macrophage cells (BoMac) . Prior to inactivation, the virus was titrated, by Reed and Müench method, in order to incubate the BoMac cells with a quantity of virus representing a multiplicity of infection of 10. Extracellular fluorescence was quenched with Trypan Blue. The ability of the antibodies to opsonize viral particles was analyzed by flow cytometry, using a FACSCalibur (Becton Dickinson, San Jose, CA) and CellQuest software. Results were expressed as % of BoMac cells incorporating FITC-iFMDV after incubation with sera. The cut-off was calculated as the mean percentage of BoMac cells incorporating FITC-iFMDV incubated with five negative sera, plus 2 SD.
Percentage of FMDV neutralization
The percentage of virus neutralized by serum from immunized cattle, at 38 and 44dpv (upon 2 and 3 peptide doses, respectively) was measured as previously describe  with minor modifications. Briefly, a 1/8 serum dilution was incubated with different 10-fold dilutions of infective FMDV (1000 to 1 TCID50), and the infective virus recovered was determined by a TCID50 assay. Titers were expressed as the % of the initial virus neutralized upon incubation. Significant differences are indicate as *** (p<0.001).
Serum Neutralization titer
Serum samples were examined for anti-FMDV neutralizing antibodies (fixed virus, variable serum) as described before . Briefly, serial dilutions of inactivated sera were incubated for 1 h at 37°C with 100 TCID50 of infective FMDV. Then virus-serum mixtures were seeded on BHK-21 monolayers. After 40 min at 37°C, fresh medium supplemented with 2% FCS was added to the cells that were incubated at 37°C under 5% CO2. Cytopathic effects were observed after 48 h. Titers of virus neutralizing antibodies were expressed as log10 of the reciprocal of the serum dilution which neutralize 50% of 100 FMDV DICT50.
Peripheral blood mononuclear cells (PBMC) were obtained from cattle as described  and 2x107 cell/ml were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) 3 μM in PBS for 15 min at 37°C. Labeled cells were added to 96-well plate (5 x 105 cell/well) containing (i) 5μg/ml iFMDV, (ii) 50μg/ml of peptides B2T, B4T or T, and (iii) 5μg/ml Concanavalin A (Sigma–Aldrich, S t. Louis, MO). Cells were incubated at 37°C in 5% CO2 atmosphere 5 days, then 0.2% paraformaldehyde was added and cell proliferation was analyzed by flow cytometry. Results were expressed as %CFSE proliferation. The cut-off was the mean % CFSE proliferation in wells without stimuli plus 2 SD.
PBMC were cultured with 50μg/ml of B2T, B4T or T peptides for 72 h. Supernatants were analyzed using ELISA as described previously . Briefly, plates were coated with a mAb against- IFN-γ (kindly donated by Dr. L. Babiuk). Samples and recombinant IFN-γ standard (Serotec, UK) were added and IFN was detected using rabbit polyclonal anti-IFN-γ antibodies. After incubation, biotinylated goat anti-rabbit IgG antibody was added and then, HRP-conjugated streptavidin (KPL, USA). Plates were washed, incubated with (OPD)-H2O2 and read at 492nm. IFN-γ concentration was calculated from interpolation of data in the standard curve.
RNA extraction and PCR amplification of viral RNA
Samples were obtained from lesions of the disease on tongue and viral RNA was extracted using Trizol (Invitrogen) and used as template of a reverse transcription reaction performed with random primers. The resulting cDNA was used to carry out PCR amplification of, on the one hand, the FMDV 3D polymerase gene (primers: GK2 -antisense- CTAGACCGTGTTGGTGGGTT and GK7 -sense- CCGACCACCACGGTGTTTTCG) and of host 18S ribosomal RNA as internal control. Amplicons of 380 bp and 480 bp were obtained for 3D viral polymerase and host rRNA, respectively. On the order hand, PCR amplification of FMDV VP1 gene (primers: P12B -antisense- TTCGAAGTACCAGGGTTTGGC and ALFA1 -sense- CTCGTTCATCATGGACAGATT). Amplicons of 780pb were obtained and direct sequencing of the PCR products was performed by capillary electophoresis.
B2T and B4T design and synthesis
B2T and B4T were designed as novel vaccine candidates as described in Fig 1. These new formulations displayed either two or four copies of the B-cell epitopes VP1 [135–160]  together with a single copy of the bovine T-cell epitope VP1 [21–40] [11, 12] both derived from FMDV O1 Campos and were produced as described in 2.1.
B2T and B4T induce anti-peptide and FMDV specific antibodies
All animals inoculated with either B2T or B4T constructs developed specific and pronounced anti-peptide as well as anti-FMDV total IgG and IgG1 responses (Fig 2A, 2B and 2C), including animal 263 that showed increased levels of antibodies against FMDV after the third peptide dose (Fig 2B). Bovines 257 and 264 (B2T) presented high levels of IgG1 only after the second peptide dose (Fig 2C). At 44dpv, high anti-FMDV IgGs titers were detected in all animals (3.9±0.1 and 4.0±0.2 in B2T and B4T groups, respectively) (Fig 2B). Low levels of specific FMDV IgG2 were detected in both B2T and B4T groups with average antibody titers of 1.36±0.25 and 1.5±0.4, respectively (data not shown).
Animals were immunized on day 0, 18 and 38 (arrow) with B2T or B4T vaccine. (A) Kinetics of anti-peptide serum antibodies. (B, C) Kinetics of total IgG anti-FMDV and IgG1 anti-FMDV serum antibodies. Titers were calculated as log 10 of last reciprocal dilution above cut-off. (D) FMDV-specific mucosal IgG1 and IgA response. Nasal swabs were collected at 28dpv. Each point represents the nasal IgG1 or IgA anti-FMDV antibody titers (log10) of each animal. Cut-off was established as the mean value of mock vaccinated animals plus twice the standard deviation value.
FMDV-specific mucosal immunity
Animals from B2T and B4T groups exhibited also high levels of anti-FMDV IgG1 in nasal secretions at 28dpv, with exception of bovine 263 that only showed high IgG1 titers (7±1.0) after the third immunization. When IgA was measured in nasal secretions, 1 out of 5 animals in the B2T and 2 out of 5 in the B4T group, showed anti-FMDV IgA titers (Fig 2D) indicating that these novel peptide constructs were able to induce not only systemic but also local mucosal immunity.
Analysis of the opsonic capacity of the sera
The importance of antibodies in FMD protection might be related to their ability to opsonize rather than to neutralize viral particles [27, 28]. The average values of opsono-phagocytosis of sera collected at the day of challenge (44dpv) were 24±11% in B2T group, 31±14% in B4T group and 35±6% in cattle immunized with an FMDV commercial vaccine (Fig 3). These values were significantly higher than those of sera before immunization (negative controls; 15±2%). In the B2T group, sera from 2 of the 5 animals showed significant responses, while 4 of the 5 animals in the B4T group responded positively. In general, a trend towards higher opsonization values was observed in sera from animals immunized with the B4T construct, similarly as the ones induced with the commercial vaccine.
Results are expressed as the % of BoMac cells incorporating FITC-virus after incubation with sera from B2T, B4T, commercial vaccine or negative sera. Each bar represents the mean of opsonising capacity of sera + SEM. * and *** represents significant differences (p<0.05 or p<0.001, respectivelly) compared to opsonising capacity of negative sera (from day 0). The cut-off was calculated as the mean percentage of cells incubated with FITC-FMDV- and sera free of FMDV antibodies plus 2 SD. The number of positive sera/ the total number of sera analyzed are indicated above bars.
Analysis of the neutralizing capacity of the sera
We first determined the percentage of virus neutralized by a 1/8 dilution of sera from immunized cattle. At 38dpv, the average values were 29±3% in animals from B2T group and 26±10% in those from B4T group (Fig 4). However, when a virus neutralization test (VNT) was performed to detect the antibody dilution capable of neutralizing a fixed amount of virus, no serum sample at 38dpv was able to neutralize 100 TCID50 of FMDV O1Campos (VNT<0.9) (data not shown), supporting a weak induction of neutralizing antibodies in these animals. Taking into account these results, a third dose of each construct was administered to the calves that resulted in a significant increase at 44 dpv in the neutralizing capacity of sera (p<0.001) (Fig 4), being the percentages of neutralization 44±3% and 46±3% for animals in B2T and B4T groups, respectively. Remarkably, these values were similar to those of a bovine immunized with a single dose of commercial vaccine (45±10%). Interestingly, all animals were also positive for VNT, being the mean titers of antibodies in animals from B4T group (1.7±0.3) significantly higher (p<0.05) than those from B2T group (1.4±0.1). Again, these VNT values were similar to those observed in animals vaccinated with commercial vaccine (1.9±0.6) (Table 1).
(A) % of virus neutralized by serum from cattle immunized with B2T, B4T or a single dose of conventional vaccine, at 38 and 44dpv (upon 2 and 3 doses of peptide, respectively). A 1/8 serum dilution was incubated with different doses of FMDV O1 Campos, and the infective virus recovered was determined by a TCID50 assay. Titers are expressed as the % of the initial virus neutralized upon incubation. Significant differences from dose are indicate as ***(p<0.001).
Specific cellular immune response and IFN-γ release in vaccinated animals
Before challenge, at 44dpv, specific in vitro lymphoproliferations were conducted using several stimuli. Significant values of proliferation (% CFSE proliferation ≥4) to the peptide used for immunization were found in 4 out of 5 animals of B2T group and in all the five animals from B4T group (Table 2A). Responses to dendrimers (B2T or B4T) not used for immunization were similar to those achieved with the immunizing peptide while the number of animals that recognized the T cell peptide alone was lower. In group B2T, PBMC from bovines 256 and 257 significantly proliferated in response to the T peptide and animal 267 showed no response to any stimulus. In group B4T only cells from bovine 263 and 271 proliferated when were stimulated with the T peptide. PBMC from a negative control animal (Table 2A) and from all bovines at day 0 did not respond to any peptide (data not shown).
Levels of IFN-γ secreted by PBMCs from immunized animals were also determined at 44dpv (Table 2B). Positive IFN-γ responses were found in 4 out of 5 animals of B2T group and in all animals of B4T group following Ag-B2T stimulation. Whereas, Ag-B4T induced IFN-γ secretion in PBMCs from 3 animals of B2T group and from 4 animals belonging to the B4T group. In group B2T, only cells from bovine 256 and 257 secreted IFN-γ when they were stimulated with the T peptide, whereas only PBMCs from bovine 263 from group B4T animals recognized this peptide for IFN-γ secretion.
B2T and B4T protect cattle against viral challenge
Subsequently, all animals were challenged at 44dpv by nasal instillation with infective FMDV O1 Campos and protection was measured by monitoring clinical signs in animals after challenge. As shown in Table 3, the three negative control animals showed typical FMDV lesions while, remarkably, all B2T and B4T vaccinated bovines were protected against podal generalization. Only animals 257 and 264, belonging to B2T group showed a single vesicle in tongue at 7dpc even though they were PPG. These two lesions were positive for FMDV RNA detection by RT-PCR (Table 3).
A sequence analysis of the capsid coding region corresponding to the B-cell epitope of the FMDV RNA directly recovered from the lesions were performed. A synonymous mutation was found in animal 257 (B-cell epitope unchanged) and a nonsynonymous mutation in animal 264. In the latter, a valine was replace by an alanine at the 144 amino acid position, within B-cell epitope (-1 RGD position)
Synthetic peptides corresponding to protective B- and T-cell epitopes are considered good candidates for safer and more effective FMD vaccines . The group led by Dr. Mowat reported in 1986 for the first and only time the successful protection against FMDV in cattle . The linear peptide spanning the VP1 regions (residues 141 to 158 and 200 to 213 of the serotype O1, 40 residue peptide) synthesized collinearly, induces a neutralizing antibody response and protected two out of three animals vaccinated with a single dose of peptide and three all animals with a revaccination with low dose at 32 days post-vaccination. Nevertheless, all subsequent studies of vaccination with linear peptides on this important FMDV natural host and with a larger number of animals resulted in partial  or null protection . Multiple presentation of the B-cell epitope has been proven advantageous over simpler arrangements when eliciting humoral and cellular immune responses . Indeed, a dendrimer peptide displaying four copies of the B cell epitope VP1 (136–154) and one copy of the T-cell epitope 3A(21–40) from type C FMDV, assembled using a thioether conjugation chemistry protects against homologous FMDV challenge in swine . Recently, Blanco et al (2016) reported full protection in pigs vaccinated with the analogous dendrimer B2T with sequences from type O FMDV O-UKG assembled using maleimide linkage, an advantageous conjugation chemistry in terms of production simplicity, with positive impact in costs and adaptability . Another important point to highlight is that synthetic peptide with low immunogenicity can promote selection of viral escape mutants as they frequently include a single or a very limited number of B cell neutralizing sites . However, the use of dendrimeric peptides can overcome this potential limitation as these structures allow the incorporation of different B cell epitopes into a single molecule.
Hence, the present report aimed to assess the effectiveness of novel maleimide-conjugated dendrimeric peptides in cattle in affording protective immune responses to FMDV. These new constructs displayed either two or four copies of the B-cell epitope (135–160 from VP1, type O FMDV O1 Campos) and one T-cell epitope 21–40 from VP1 (named B2T and B4T, respectively). Our results indicate that these two dendrimers afforded 100% PPG to viral challenge.
When compared with the immune response evoked by analogous dendrimers in swine, the main difference observed in bovine is the need for a third immunization to detect consistent levels of neutralizing antibodies. It is possible that the immunogenicity in cattle can be increased by using other adjuvants or different amounts of peptide in the vaccine. Moreover, after a third dose of vaccine containing a low amount of peptide, we observed an enhancement of the IgG affinity rather than an increase on the level of IgGs. The third immunization could have resulted into a more stringent selection of higher affinity B cells, and the emergence of effective neutralizing antibodies.
In addition, pigs vaccinated with dendrimer B(OUK)2T3A elicited increased levels of virus-specific IgG1 and IgG2 relative to those of animals vaccinated with B(OUK)4T3A, while in this study immunization of cattle with B2T or B4T elicited similar levels of total anti-peptide and anti-FMDV antibodies, as well as high levels of specific IgG1 against the virus. Further experiments are required to understand the low levels of IgG2 detected in this study; noteworthy, the same nomenclature for subclasses among different species often leads to the mistaken believe that these subclasses are homologous and have the same functions .
Induction of high levels of FMDV-specific IgG1 subtype in serum has been related to protection in conventionally vaccinated cattle, even at low levels of total IgG . Furthermore, differential IgG1/IgG2 ratios have been associated with protection in animals with low VNT titers , as well as in bovines immunized with linear synthetic peptides . Bovine IgG1 is involved in neutralization and also in pathogen opsonization.
Macrophages have a high phagocytic capacity and they can engulf and destroy FMDV, especially if it is opsonized by specific antibodies [27, 34]. At day 44dpv, high opsonophagocytosis levels were found in sera from animals vaccinated with the dendrimeric constructs, with values similar to those from bovines vaccinated with a commercial vaccine.
Animals of the B4T group were not only protected from podal generalization but also did not develop lesions in tongue or mouth, near the viral instillation point, which could be related with the trend towards higher opsonization and VNT values observed in sera of these animals when compared with those immunized with B2T. One of the two B2T-vaccinated cattle that developed tongue lesions (animal 264) showed an amino acid substitution in the region corresponding to the B epitope, which could reflect the selection of a scape mutant in this partially protected animal.
In animals immunized with B4T, VNT titers were similar to those of bovines immunized with the commercial vaccine (approved by SENASA for vaccination in Argentina) and associated with Expected Percentage of Protection (EPP) of 80 percent. The EPP estimates the likelihood that cattle would be protected against a challenge of 10.000 bovine infective doses after vaccination . Bovine 253 that elicited low neutralizing antibodies levels (1.2) resulted protected upon challenge, which could be associated with the presence of high IgG1 levels in its serum, in accordance with the results published by Capozzo et al .
Importantly, all antibodies induced by the dendrimeric peptides used in this study allowed differentiation of infected from vaccinated animals, since they did not react against the NS protein 3ABC (data not shown), which is the standard diagnostic tool for FMDV replication .
It is plausible that the improved immunogenicity of constructs with four versus two epitopes is based on positing repetitive antigens that induce direct cross-linking of surface Ig receptors in immature B cells  or that epitope multimerization promotes antigen internalization by dendritic cells or other antigen presenting cells . In any case, B4T and B2T efficiency in cattle differs from that described for their counterparts in pigs  and further experiments are needed to understand these differences among dendrimers that display different B and T cell sequences and target different species. The T-cell epitope VP1 (21–40), previously identified in bovine  was incorporated to B2T and B4T due to the lack of experimental evidence supporting recognition as a T-helper epitope in cattle of the 3A (21–35) epitope included in the dendrimers that successfully protected swine .
FMDV usually gains entry through the respiratory tract of cattle . There are a number of mechanisms that have been proposed to explain the production of secretory antibodies after immunization with antigen. One of the mechanisms proposed is the direct diffusion of soluble or phagocytosed antigens to mucosal-associated lymphatic tissues (MALT); other is the migration of activated antigen-presenting cells to MALT from the lymph node that drains the inoculation site.[39, 40]. However, in mucosa, regulation of IgA and IgG1 production in cattle is not completely understood and further studies should be aimed at establishing the association between these immunoglobulins and protection. In animals belonging to B2T and B4T groups, IgG1 specific against FMDV was increased in nasal secretions, suggesting some role of this immunoglobulin in protection; however, only some animals showed an increased IgA in nasal secretions. It is tentative to speculate the possible implications of both IgG1 and IgA immunoglobulins against viral entry in mucosal tissues.
Animal to animal variation was observed in both the lymphoproliferation and IFN-γ secretion assays, as reported in previous studies with peptide-immunized cattle . Interestingly, B2T and B4T peptides evoked similar consistent T cell responses, being recognized in vitro by lymphocytes from most of the immunized cattle in the proliferation assay, and from all animals in the IFN-γ production assay. The T-cell epitope VP1(21–40] alone was recognized by a lower number of peptide vaccinated bovines, and it was not as efficient inducing cellular responses as B2T and B4T peptides, which could be due to a better presentation/processing of the dendrimers in the in vitro assays used. On the other hand, it has been described that the 135–160 region of VP1 there is recognized as a T-cell epitope in bovine (135–144) .
The number of bovines included in this pilot study was similar to those in other introductory studies on vaccine candidates [4, 6, 42, 43], although we were aware that these numbers are not enough for statistical demonstration .
In conclusion, our results support that immunization of cattle with dendrimeric peptides such as B2T or B4T can evoke humoral immune responses similar to those induced by commercial vaccines. More importantly, they were able to elicit protective responses preventing clinical disease. These results supported our new dendrimeric peptide constructs as a promising candidate for peptide subunit DIVA vaccines against FMDV in cattle.
We thank Dr N. Fondevilla and O. Zabal for his invaluable work in the BSL-4 OIE facilities at the CICVyA–INTA and to Mr D. Soraire, J. Leiva, D. Romero, C. Fioroni, D. Franco, Dr V. Maldonado for their help in handling and caring for experimental animals. We also thank to Mrs P.Angeletti for her excellent technical and administrative assistance in our laboratory and Dr C. Perez-Beascoechea for preparing and providing the infectious FMDV.
- 1. Barteling SJ, Cassim NI. Very fast (and safe) inactivation of foot-and-mouth disease virus and enteroviruses by a combination of binary ethyleneimine and formaldehyde. Developments in biologicals. 2004;119:449–55. pmid:15742659.
- 2. Diaz-San Segundo F, Medina GN, Stenfeldt C, Arzt J, de Los Santos T. Foot-and-mouth disease vaccines. Vet Microbiol. 2016. pmid:28040311.
- 3. Cubillos C, de la Torre BG, Jakab A, Clementi G, Borras E, Barcena J, et al. Enhanced mucosal immunoglobulin A response and solid protection against foot-and-mouth disease virus challenge induced by a novel dendrimeric peptide. J Virol. 2008;82(14):7223–30. pmid:18448530.
- 4. Blanco E, Guerra B, de la Torre BG, Defaus S, Dekker A, Andreu D, et al. Full protection of swine against foot-and-mouth disease by a bivalent B-cell epitope dendrimer peptide. Antiviral Res. 2016;129:74–80. pmid:26956030.
- 5. Bittle JL, Houghten RA, Alexander H, Shinnick TM, Sutcliffe JG, Lerner RA, et al. Protection against foot-and-mouth disease by immunization with a chemically synthesized peptide predicted from the viral nucleotide sequence. Nature. 1982;298(5869):30–3. pmid:7045684.
- 6. DiMarchi R, Brooke G, Gale C, Cracknell V, Doel T, Mowat N. Protection of cattle against foot-and-mouth disease by a synthetic peptide. Science. 1986;232(4750):639–41. pmid:3008333.
- 7. Taboga O, Tami C, Carrillo E, Nunez JI, Rodriguez A, Saiz JC, et al. A large-scale evaluation of peptide vaccines against foot-and-mouth disease: lack of solid protection in cattle and isolation of escape mutants. J Virol. 1997;71(4):2606–14. pmid:9060612.
- 8. Blanco E, Cubillos C, Moreno N, Barcena J, de la Torre BG, Andreu D, et al. B epitope multiplicity and B/T epitope orientation influence immunogenicity of foot-and-mouth disease peptide vaccines. Clin Dev Immunol. 2013;2013:475960. pmid:24454475.
- 9. Sadler K, Tam JP. Peptide dendrimers: applications and synthesis. J Biotechnol. 2002;90(3–4):195–229. pmid:12071226.
- 10. Heegaard PM, Boas U, Sorensen NS. Dendrimers for vaccine and immunostimulatory uses. A review. Bioconjugate chemistry. 2010;21(3):405–18. pmid:19886668.
- 11. Collen T, Dimarchi R, Doel TR. A T cell epitope in VP1 of foot-and-mouth disease virus is immunodominant for vaccinated cattle. J Immunol. 1991;146(2):749–55. pmid:1702816.
- 12. Glass EJ, Oliver RA, Collen T, Doel TR, Dimarchi R, Spooner RL. MHC class II restricted recognition of FMDV peptides by bovine T cells. Immunology. 1991;74(4):594–9. pmid:1664415.
- 13. Monso M, de la Torre BG, Blanco E, Moreno N, Andreu D. Influence of conjugation chemistry and B epitope orientation on the immune response of branched peptide antigens. Bioconjugate chemistry. 2013;24(4):578–85. pmid:23458489.
- 14. Pega J, Bucafusco D, Di Giacomo S, Schammas JM, Malacari D, Capozzo AV, et al. Early adaptive immune responses in the respiratory tract of foot-and-mouth disease virus-infected cattle. J Virol. 2013;87(5):2489–95. pmid:23255811.
- 15. Maradei E, La Torre J, Robiolo B, Esteves J, Seki C, Pedemonte A, et al. Updating of the correlation between lpELISA titers and protection from virus challenge for the assessment of the potency of polyvalent aphtovirus vaccines in Argentina. Vaccine. 2008;26(51):6577–86. pmid:18835312.
- 16. Orsel K, Bouma A. The effect of foot-and-mouth disease (FMD) vaccination on virus transmission and the significance for the field. Can Vet J. 2009;50(10):1059–63. pmid:20046605.
- 17. Orsel K, Dekker A, Bouma A, Stegeman JA, de Jong MC. Vaccination against foot and mouth disease reduces virus transmission in groups of calves. Vaccine. 2005;23(41):4887–94. pmid:16002192.
- 18. Sellers RF, Donaldson AI, Herniman KA. Foot-and-mouth virus. Lancet. 1971;1(7711):1238. pmid:4103095.
- 19. (OIE) WOfAH. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. http://wwwoieint/fileadmin/Home/eng/Health_standards/tahm/20108_FMDpdf. 2017.
- 20. Quattrocchi V, Pappalardo JS, Langellotti C, Smitsaart E, Fondevila N, Zamorano P. Early protection against foot-and-mouth disease virus in cattle using an inactivated vaccine formulated with Montanide ESSAI IMS D 12802 VG PR adjuvant. Vaccine. 2014;32(19):2167–72. pmid:24631088.
- 21. Quattrocchi V, Soria I, Langellotti CA, Gnazzo V, Gammella M, Moore DP, et al. A DNA Vaccine Formulated with Chemical Adjuvant Provides Partial Protection against Bovine Herpes Virus Infection in Cattle. Frontiers in immunology. 2017;8:37. pmid:28179907.
- 22. Huber VC, Lynch JM, Bucher DJ, Le J, Metzger DW. Fc receptor-mediated phagocytosis makes a significant contribution to clearance of influenza virus infections. J Immunol. 2001;166(12):7381–8. pmid:11390489.
- 23. Stabel JR, Stabel TJ. Immortalization and characterization of bovine peritoneal macrophages transfected with SV40 plasmid DNA. Vet Immunol Immunopathol. 1995;45(3–4):211–20. pmid:7676607.
- 24. Tekleghiorghis T, Weerdmeester K, van Hemert-Kluitenberg F, Moormann RJ, Dekker A. Comparison of test methodologies for foot-and-mouth disease virus serotype A vaccine matching. Clinical and vaccine immunology: CVI. 2014;21(5):674–83. pmid:24623625.
- 25. Romera SA, Puntel M, Quattrocchi V, Del Medico Zajac P, Zamorano P, Blanco Viera J, et al. Protection induced by a glycoprotein E-deleted bovine herpesvirus type 1 marker strain used either as an inactivated or live attenuated vaccine in cattle. BMC veterinary research. 2014;10:8. pmid:24401205.
- 26. Zamorano PI, Wigdorovitz A, Perez Filgueira DM, Escribano JM, Sadir AM, Borca MV. Induction of anti foot and mouth disease virus T and B cell responses in cattle immunized with a peptide representing ten amino acids of VP1. Vaccine. 1998;16(6):558–63. pmid:9569465.
- 27. McCullough KC, Parkinson D, Crowther JR. Opsonization-enhanced phagocytosis of foot-and-mouth disease virus. Immunology. 1988;65(2):187–91. pmid:2847979.
- 28. Law M, Hangartner L. Antibodies against viruses: passive and active immunization. Current opinion in immunology. 2008;20(4):486–92. pmid:18577455.
- 29. Orsel K, de Jong MC, Bouma A, Stegeman JA, Dekker A. Foot and mouth disease virus transmission among vaccinated pigs after exposure to virus shedding pigs. Vaccine. 2007;25(34):6381–91. pmid:17658199.
- 30. Rodriguez LL, Barrera J, Kramer E, Lubroth J, Brown F, Golde WT. A synthetic peptide containing the consensus sequence of the G-H loop region of foot-and-mouth disease virus type-O VP1 and a promiscuous T-helper epitope induces peptide-specific antibodies but fails to protect cattle against viral challenge. Vaccine. 2003;21(25–26):3751–6. pmid:12922108.
- 31. Pastoret P-P. IMMUNOLOGY OF CATTLE In: Griebel P, Bazin H, Govaerts A, editors. Handbook of Vertebrate Immunology. San Diego: Academic Press; 1998. p. 439–84.
- 32. Capozzo AV, Periolo OH, Robiolo B, Seki C, La Torre JL, Grigera PR. Total and isotype humoral responses in cattle vaccinated with foot and mouth disease virus (FMDV) immunogen produced either in bovine tongue tissue or in BHK-21 cell suspension cultures. Vaccine. 1997;15(6–7):624–30. pmid:9178462.
- 33. Lavoria MA, Di-Giacomo S, Bucafusco D, Franco-Mahecha OL, Perez-Filgueira DM, Capozzo AV. Avidity and subtyping of specific antibodies applied to the indirect assessment of heterologous protection against Foot-and-Mouth Disease Virus in cattle. Vaccine. 2012;30(48):6845–50. pmid:23000129.
- 34. McCullough KC, De Simone F, Brocchi E, Capucci L, Crowther JR, Kihm U. Protective immune response against foot-and-mouth disease. J Virol. 1992;66(4):1835–40. pmid:1312607.
- 35. Brocchi E, Bergmann IE, Dekker A, Paton DJ, Sammin DJ, Greiner M, et al. Comparative evaluation of six ELISAs for the detection of antibodies to the non-structural proteins of foot-and-mouth disease virus. Vaccine. 2006;24(47–48):6966–79. pmid:16753241.
- 36. Bachmann MF, Rohrer UH, Kundig TM, Burki K, Hengartner H, Zinkernagel RM. The influence of antigen organization on B cell responsiveness. Science. 1993;262(5138):1448–51. pmid:8248784.
- 37. Zinkernagel RM. On natural and artificial vaccinations. Annu Rev Immunol. 2003;21:515–46. pmid:12500980.
- 38. Alexandersen S, Mowat N. Foot-and-mouth disease: host range and pathogenesis. Current topics in microbiology and immunology. 2005;288:9–42. pmid:15648173.
- 39. Bouvet JP, Decroix N, Pamonsinlapatham P. Stimulation of local antibody production: parenteral or mucosal vaccination? Trends in immunology. 2002;23(4):209–13. pmid:11923116.
- 40. Pega J, Di Giacomo S, Bucafusco D, Schammas JM, Malacari D, Barrionuevo F, et al. Systemic Foot-and-Mouth Disease Vaccination in Cattle Promotes Specific Antibody-Secreting Cells at the Respiratory Tract and Triggers Local Anamnestic Responses upon Aerosol Infection. J Virol. 2015;89(18):9581–90. pmid:26157128.
- 41. Zamorano P, Wigdorovitz A, Chaher MT, Fernandez FM, Carrillo C, Marcovecchio FE, et al. Recognition of B and T cell epitopes by cattle immunized with a synthetic peptide containing the major immunogenic site of VP1 FMDV 01 Campos. Virology. 1994;201(2):383–7. pmid:8184548.
- 42. Porta C, Xu X, Loureiro S, Paramasivam S, Ren J, Al-Khalil T, et al. Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity. J Virol Methods. 2013;187(2):406–12. pmid:23174161.
- 43. Moraes MP, Mayr GA, Mason PW, Grubman MJ. Early protection against homologous challenge after a single dose of replication-defective human adenovirus type 5 expressing capsid proteins of foot-and-mouth disease virus (FMDV) strain A24. Vaccine. 2002;20(11–12):1631–9. pmid:11858872.

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