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Category: Architecture
Wainscot Panelling and Panelled Walls
Posted in Oct 2019 by Andrew Petherick
The History of Panelling
Wainscot Panelling, or Wainscoting, is a style of panelling which developed off the back of early panelling methods.
In it's first instance, wood panelling was developed as a practicality. It provided insulation and covered up any damp that infiltrated cold stone walls. Yet, it was soon recognised as a decorative technique, adding detail and warmth to a room. In the 13th century, Henry III imported wood from Norway and used it to line rooms at Windsor Castle. As time went on, decorative panelling turned into a fine art.
Linen-fold panelling was a decorated and embellished style that became popular in the 15th century. Boiserie panelling, which is ornate and intricately carved, became favored in French interior design in the late 17th century. This type of panelling lined walls, doors, cupboards and shelves and great examples of this style can be found at Hampton Court Palace in Richmond.
During the English Renaissance, wood panelling became simpler in design. In grand houses, applied pilasters appeared to provide an architrave which elegantly concealed the join between panels. Applied pilasters then became a common feature of classical Georgian interior architecture, punctuating walls to emphasise window positions and bring structure to a large space.
Boiserie from the Hôtel de Cabris, Grasse,ca. 1774, with later additions
Traditional Linenfold Panelling
An example of 18th century, Georgian style Paneling
Furthermore, during the 18th century, a new panelling style came into fruition: Wainscot panelling. Product of Danish Wainscot Oak, this panelling was charactersied by only covering the lower section of a wall, leaving a dado above. Wainscot Oak produced large, knot-free boards that were attractive and easy to work with, making it more favorable than oak grown in Britain.
Wainscoting is still a popular panelling style today.
Simple Wainscot Panelling in a hallway
Wainscot Paneling in a Bathroom
Panelling in Your Home
When decorating your country home, it is always important to consider it's period and history; this should then be a used to influence your internal architecture and decoration style. For example, in Victorian houses, it would be acceptable to have Edwardian panelling. However, it would not make stylistic sense to have Victorian panelling in an Edwardian house as the chronology would be backwards. Panelling with sunk framed squares, or rectangles, was popular in the 16th and 17th century and is particularly appropriate for restoring a country manor house. The period of a building influences it proportions both externally and internally, and such considerations are central to our design process at Artichoke.
Nowadays, it is possible to panel walls in a variety of beautiful woods from across the world. Panelling can be made to suit both traditional and modern interior decoration. If you are interested in painted panelling, a more contemporary style, then a hard wood such as Poplar can be an appropriate choice. For panelling that showcases the wood grain, Sapele or soft wood, such as European Red Wood, can be effective. For traditional paneling styles, mahogany, walnut and oak are classic. Using stains and polishes will age and give character to any wood. In softer rooms, such as bedrooms, fabric panels can be used to add texture and grandeur. In formal rooms, adding marquetry into panels can be striking and ornate.
Wainscoting lends well to darker, smaller rooms, where full walled panelling may be too oppressive. Leaving the top section of a wall to be painted or papered, offers an opportunity for color and pattern alongside a traditional panel.
An example of full walled, Georgian style paneling in stained and hand-polished French Walnut, designed and made by Artichoke. See more of this project here:
Panelling details by Artichoke
If you own a listed property, it may be appropriate to speak to your conservation officer or take advice from Historic England. In cases of preserving and renovating authentic panelling, working with a specialist is recommended.
As experts in historical architecture and period joinery, Artichoke can offer trusted, bespoke panelling design. Working closely with our suppliers, we have access to some of Worlds most beautiful timbers and veneers.
Painted Wainscoting in a traditionally styled bathroom.
To discuss your project with Artichoke, email us at newprojects@artichoke.co.uk
Posted in ArchitectureTagged decorative paneling, decorative panelling, linenfold paneling, linenfold panelling, paneled rooms, paneled walls, panelled rooms, panelled walls, wainscot paneling, wainscot panellingLeave a Comment on Wainscot Panelling and Panelled Walls
Building a Country House? Speak to Tim Moulding First
Posted in Sep 2019 by Andrew Petherick
Building a country house is a dream for many, and those with the opportunity will naturally approach it with great enthusiasm.
Enthusiasm is a wonderful thing, but it can sometimes cloud judgement. When you are eager to get going, it is easy to rush ahead and glaze over more menial but important decisions and plans. In most cases, we learn from these mistakes through experience. However, when building a country house, mistakes can be expensive and in the worst case scenario, irreversible. You want to get it right first time.
Tim Moulding, MD of the South's leading traditional building companies, R. Moulding & Co
The First Step to Building a Country House? Ask Someone with Experience.
Building consultant, Tim Moulding, now offers Country House Building Consultancy, a sensible first step for anyone with more enthusiasm than experience in the creation of their country home.
One of Tim's continual frustrations is the inheritance of poor process. As a builder, it is rare for him to be given the opportunity to influence the design or method of a country house project from its inception. Too often, a builder is expected to deliver a project as it is handed to them, which is typically after a project has confirmed planning and therefore cannot be altered. Like Tim, we think these scenarios are missed opportunities. A great deal of frustration, delay and rising costs can be avoided if specialists are consulted before the detail is locked down.
Stockton House in Wiltshire, renovated by R Moulding and Co (affectionately known as Mouldings)
Tim has more experience than most in the successful delivery of country house projects. As well as being a consultant, he is also the eighth-generation owner and Managing Director of the leading traditional building company, R. Moulding & Co (affectionately known as Mouldings). For the past 30 years he has led his team in the delivery of over 70 residential building projects ranging from £500k to £15m in value. This experience has given Tim an intimate understanding of the building process specific to residential country houses.
Like Artichoke, Tim has realised that there is a gap in the market for a service that provides advice to anyone purchasing, or who owns a country home, at the onset of a major building project. Tim is now offering his expertise, independently of his building firm, providing sound guidance towards the delivery of a successful building project before it's too late. At Artichoke, we see the affects of lack of consultancy first hand; often clients have misplaced the joinery design of their country home. It is then a big task to pick up the pieces and the client ends up to paying twice for a design (as this previous artichoke blog explores further).
Question the Key Elements of the Building Process
In the context of country house restoration or new builds, Tim believes the process can be positively influenced by a number of factors. Examining a project from the outset can hugely improve the efficiency and cost of designing and building a country house, ultimately improving the owner's enjoyment of the process.
His consultancy is wide-ranging and explores everything from evaluating the experience of designers, to accessing the approach a construction team should take. When the answers aren't binary, making decisions with the input of expertise can massively improve the efficiency and success of a building project. Tim's considerable experience in this field may be the ideal remedy to any doubts you may have.
For more information on Country House Building Consultants, visit Tim's website.
Posted in ArchitectureTagged building a country house, mouldings builders, r moulding builders, tim mouldingLeave a Comment on Building a Country House? Speak to Tim Moulding First
Designing into the Elizabethan Country House
Posted in Apr 2019 by Andrew Petherick
Many Artichoke projects tend to focus on Georgian and Victorian period country houses, with the occasional sojourn to the later Edwardian or to earlier Jacobean and Elizabethan periods. It is fascinating to see how styles of architecture and interior detailing evolve through the English country house. The entire post Norman history is deeply illustrated through architecture.
One architectural style we find particularly enchanting is the Elizabethan country house. Our first major country house project, over 20 years ago, was Parnham House in Dorset, one of Britain's finest examples of Elizabethan architecture. We were commissioned by the owners to design various rooms including both kitchens, a private dressing room and a minstrell's gallery, all made in our workshops in Somerset. With great sadness, the house was extensively damaged by fire in 2018 and it is now on the market.
Parnham House, built during the reign of Elizabeth I and reconfigured by John Nash in the 19th century.
Elizabethan Country House Architecture
The Elizabethan period (1560-1600) was a period of design transition in architecture. England's understanding of the Italian Renaissance was just coming to the forefront, spurned on by Henry VIII's numerous contacts with Italy before breaking with Rome. When Elizabeth took up her reign, the country's economy started to improve following years of recklessness from Henry. A focus back into farming created more money for wider groups of people across England, and a domestic building boom began. Many smaller houses were built and many larger manors were created, often through remodelling of earlier Tudor or Medieval homes.
An early introduction to Italian Renaissance architecture was fused with England's already well established Gothic architecture, alongside a little Dutch influence. This opulent mixture was brilliantly interpreted by English craftsmen who elevated this new hybrid style to stunning levels of romantic architectural detailing.
Breamore House in Hampshire
Houses were typically symmetrical with long galleries and formal gardens, often laid out in an 'E' pattern. The medieval hall was replaced in importance by the long gallery which became the focus for family life alongside other living areas off the gallery. Key decorative characteristics included large mullioned windows with square heads and ornamental strap work (both internal and external), a detail originating in Italy via Islamic ornament. Extensive use of rectangular timber oak panelling was also prevalent, often made even richer with the use of carved strap work ornament. Combined with ornate plaster work ceilings and carved overmantels, the affect was striking and powerful.
Creating a Design Backstory
This kaleidoscope of detail presents a challenge to joinery designers like Artichoke. It is difficult to compete with it. In the Elizabethan era, domesticity as we know it today, simply didn't exist.
For Parnham House we created a strategy, giving the furniture we designed the backstory of an Edwardian interior facelift. The kitchen design took on detail from the medieval and Tudor periods, albeit with a Edwardian twist. This approach was heavily influenced by Edwin Lutyens who took a similar approach with many of his buildings which are often Edwardian takes of medieval architecture.
The backstory is a great way to contextualise and harmonise design in an imposing period building, particularly pre Georgian designs, where domestic rooms didn't exist. A backstory gives design a single direction, ensuring the end result is sympathetic, elegant and above all for an Elizabethan house, deferent.
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English Heritage & Historic England
Posted in Mar 2019 by Andrew Petherick
Recently, there has been some confusion about the difference between English Heritage and Historic England, and in particular which organisation is now responsible for overseeing amendments to listed houses.
Until lately, English Heritage was the name of the body responsible for looking after England's historic monuments and listed buildings. Their responsibilities stretched from looking after national public monuments such as Stonehenge, to works on private listed houses. In 2015 it was decided these two quite distinct responsibilities should be separated.
The body now responsible for England's listed houses and buildings (and also the Heritage at Risk register) is called Historic England. They are a public body funded by the Government, and their role is principally to manage the National Heritage List for England, which is a database of England's designated heritage assets (such as listed houses, churches, scheduled monuments and battlefields). Therefore, anyone who owns a listed property, including many of our clients here at Artichoke, will be dealing with Historic England on matters concerning alterations to their listed property.
English Heritage, the body that used to deal with homeowner, is now a charity completely separate from the listed buildings process. Their role is now to care for hundreds of historic 'public' sites across the country, such as Hadrian's Wall, Dover Castle, Osborne House and Audley End House (below)
How does each grade of listing affect your project?
Listed private houses are essentially those considered worthy of protection owing to their architectural or historic interest, with listings separated into Grade I, Grade II* and Grade II. Regardless of the grade a house is listed at, Historic England has extra control over what changes can be made to its interior and exterior. In general, each listing covers the whole building as well as any attached structures, additions or fixtures and in many cases land or buildings which come within the surrounding land or curtilage of the building (such as barns, outbuildings etc).
As can be the case with VAT and listed houses, there is little consistency between planning districts and planning officers. Some Conservation Officers can be relaxed, while others are very particular about what would appear to be the smallest detail.
If you are using a local architect, it is often worth discussing your appointed planning office, as working with the right Conservation Officer for your project can really make a difference.
For more information on the range of project management services we offer, from design, installation and finish, please click here. As ever, do call us if you'd like to discuss this particular matter further +44 (0)1934 745270
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Carrara & the English Country House
Posted in Jun 2018 by Andrew Petherick
The interior design of many of our most treasured country houses in many ways reflects the characteristics of the English themselves. Restrained, understated, subtle and, occasionally, elegant.
These are all traits the Artichoke design team tries to inject into the kitchens and furniture we design for our client's country houses. Many of these attributes are successfully delivered through the physical form of the furniture we design, such as the period mouldings we create for each piece, the width of the door frames we design and the proportion of the furniture. Achieving elegance through form can quickly be tarnished if the materials then chosen to adorn it are not well considered.
Carrara marble grain. Understated and elegant.
Our view here at Artichoke is that if a designer creates beautifully detailed kitchen furniture, very little else needs then to be added to detract from it. This is particularly so with classical furniture where mouldings and shadow inject a wonderful flow and ripple into the face of the work. Contemporary kitchens are so often adorned with striking and flamboyant marbles because the furniture itself has little creative substance to it. By introducing a bold patterned marble, the designer is simply deflecting attention away from the fact the furniture is principally flat and lifeless.
It is inevitable that as designers and makers of kitchens and domestic areas of country houses, we have a view on which marble worktops look most appropriate in traditional environments. Despite our work being principally in English country houses, we tend not to suggest English stones for many of our kitchen worktops. While many of them are extremely hard wearing (such as slates available from Lancashire), few of them can be used for pieces such as cook's tables or islands. This is mainly due to the nature of their extraction from the rock bed. Most English stones are blasted from the quarry face with explosives, resulting in eccentric sized blocks usually no wider than two metres. By contrast, marble slabs are cut from the quarry face in huge rectangular blocks, allowing for a much greater size of slab (typically up to three metres in length). This makes marble much more practical to use in kitchen design. We explore which stones perform best for kitchen worktops in another blog 'Ideas for Kitchen Worktops'.
Honed Carrara Marble worktops and backsplash form an understated background to this Artichoke kitchen.
In the Georgian and Edwardian periods of English architecture, Carrara was the favoured marble of choice. Not only was it readily available, but its quiet and understated graining also reflected the characteristics of the English themselves. It is luxurious without being opulent and it has a more understated veining compared to other more 'vulgar' marbles. It can be seen in many of England's finest country houses such as Chatsworth. Marble Arch is built from Carrara. It has been described as the elegant workhorse of the kitchen, and it ages beautifully.
From a longevity point of view, Carrara marble is also timeless. This works well with our designs which we create to sit comfortably and elegantly into their architectural surroundings for many years. If we are to create furniture today that will be admired by future generations (in much the same way that today we admire work created in houses like Chatsworth), then it is worth remembering Carrara for your project.
To discuss your project, email the Artichoke team at newprojects@artichoke.co.uk or call on +44 (0)1934 745270.
Posted in ArchitectureTagged beautifully detailed kitchen furniture, Contemporary kitchens, country houses, edwardian interiors, english architecture, georgian kitchen design, kitchens and furniture design, makers of kitchensLeave a Comment on Carrara & the English Country House
English Country House Timbers
Up until the early 20th Century, the typical English country house was principally built from timber, stone and brick; simple when compared to the plethora of material types, fixtures and fittings available to today's architect.
Of the timber choices available to those building country houses in the 18th and 19th centuries, the most common were European Walnut, Mahogany, Russian Deal, English Oak and English Elm. These timbers had different roles to play in the make up of the English country house, with Walnut and Mahogany being favoured for the more decorative elements and Russian Deal, Elm and Oak for the more constructional. Each had their part to play.
Fast forward to today, and most clients renovating a country house are increasingly sensitive to the original materials used to build it. But are the original timber species used still available, and what are the alternatives if they are not?
European Walnut and Mahogany
Then: Until the early 1700s Walnut was by far the most popular of the decorative hardwoods for use in English country houses. It had a soft colour and an interesting grain. But access to fine quality walnut ceased after 1709 when the Great Frost, the harshest European winter for 500 years, killed off much of the walnut stock in France. This triggered English cabinet-makers to look elsewhere for alternatives, with mahogany proving the outright winner. In 1721 the British Parliament removed all import duties from timber imported into Britain from the British colonies, instantly stimulating the trade in West Indian timbers including, most importantly, mahogany. With no competition from walnut, imports of mahogany into England rose from 525 tons a year in 1740 to more than 30,000 tons in 1788. In a relatively short period of time, mahogany had become the most popular timber for luxury furniture and architectural joinery in the country houses of England.
Now: Mahogany is no longer imported from the Americas although we do have old stock on supply which is reserved for very specific country house projects and feature architectural joinery doors. The only true mahogany currently imported into the UK is African Mahogany which is lighter in colour than Brazilian or Cuban mahogany which tend to be very dark orange. African mahogany also has a slightly wilder grain pattern. Between the two, our timber of choice would always be European Walnut for its softer colouring, its figure and its provenance.
French polished Italian walnut in a library project designed by Artichoke.
Russian Deal
Then: Russian deal is a high quality softwood grown in the Baltic regions of northern Russia, typically from Archangel and Onega. It is slow grown, tall, straight and dense, and with its fine grain is ideal for making hand painted interior architectural joinery. It was considered poor for exterior use however, with Rivington's Building Construction Guide (published in 1875) declaring it unfit for work exposed to the damp shores of the UK. A more in depth piece on Russian Deal was written by us a few years ago, triggered by the BBC's adaptation of War and Peace where much of the joinery in the wonderful period locations were made of this material.
Now: Russian Deal is tough to get hold of, not because it is scarce but because the timber yards in Russia will only sell it by the boatload and the boards offered are only 1 inch thick. Specialist companies such as Artichoke are of no commercial interest to these yards. Scandinavian Redwood is the next best alternative. It's almost identical albeit being a slightly smaller tree. Artichoke uses Scandinavian Redwood in listed country house projects where organisations such as Historic England require it or we feel it will benefit the feel of the final room and character of the furniture. The grain certainly looks good when over painted, and we have recently used it when designing and making a kitchen based around the National Trust's kitchen at Lanhydrock House in Cornwall. Images from this project can be found here. The reason most furniture makers do not use Scandinavian Redwood is principally because of timber movement which can make it more unpredictable in modern homes with more aggressive heating set ups. Poplar or tulipwood is (in our mind) a more sensible choice if the project is to have a crisp hand painted finish with no grain grinning through the paint. It is resin and almost knot free and dense if you buy it from the right sources.
Separate elements of a kitchen designed by us in Siberian larch for a country house in Hampshire. The grain looks beautiful grinning through the paint surface.
Then: English oak is rot resistant, making it ideal for exterior joinery and boat building (many Queen Anne and early Georgian English country houses feature repurposed supporting oak beams which once formed part of our naval fleet). Oak's ability to resist rot, combined with its immense strength and availability, made it the perfect building material for timber framed houses, and many of the originals are still standing. Of course, much of England's ancient forests are either now protected or gone.
Now: English oak is very much readily available in the UK, although it tends to be farm or estate grown, meaning the trees have not been cared for as a commercial commodity would typically be. This makes the quality of the available material quite unpredictable and inconsistent for interior furniture such as libraries or room panelling. Our climate in Northern Europe also means English oak trees grow slowly with wild grain patterns often being a feature. English oak is also a darker shade of brown than European oak and, combined with wild grain patterns and knots, can make the furniture appear quite rustic without careful selection. At Artichoke we prefer oak from southern France where our oak trees are grown commercially and therefore managed as a crop. Buying oak for a project from the same single stand in the same area of a woodland also gives us the confidence in knowing we will receive a high quality product with a consistent honey colour throughout. Having an excellent relationship with your supply chain is vital if the work is to stand the test of time for hundreds of years.
English Oak also makes an excellent flooring material; it ages beautifully and is hard wearing. Our friends at Weldon Flooring are worth talking to if you are working in an historic or new build English country house in need of a beautiful oak floor.
This large kitchen was made from carefully selected European oak. There is a magical consistency in the grain of European Oak which would be much harder to find in English Oak.
Then: Like English Oak, English Elm is known for its rot resistant qualities making it suitable for exterior work. As one of the largest deciduous trees in the UK, it was commonly used as a building material for roof frames and supporting beams. While extremely strong with an immense ability to withstand crushing forces, it was not as popular as English oak because it tends to move and split. This is the reason that smaller English country houses, and those with agricultural links, tended to favour English Elm. In the smaller English country house, cost was a factor and you could get more out of the larger trunk.
Now: Dutch Elm disease ravaged the UK's population of English Elm between 1970 and 1990, and there are now few left, making English Elm pretty exclusive. The stock we now use for English country house work tends to be quite gnarled and rustic in appearance, so like English Oak it needs to be selected carefully. For a large English country house project we would nowadays consider European Elm, which is the same species but grown in Europe. Like oak, this material tends to be more consistent in its growth and straighter grained making it a good choice for doors and architectural joinery.
English Elm has a wild grain making it often too rustic for the grander English country house.
So in conclusion, for clients renovating or building an English country house, it is entirely possible to use timber that is appropriate to the period or to the original building materials, although its origin of source may now be different to the original.
If you are focussed on using the correct timber and materials for your country house project and are motivated to create furniture which will become an integral part of its architecture for many years to come, we'd love to hear about it!
To see some of the stunning work we have completed please click here.
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A Welcome Return to Classicism
Every 15 years or so I become fashionable. My worn jeans and faded blue shirt ensemble becomes the look for that season, and for a brief moment, I'm on trend. The downside is that I live in Somerset, so when everyone's realised what the trend is here, everyone in London's wearing something else.
As a company, Artichoke also avoids trends. Our kitchen designs are naturally classical. As designers of bespoke kitchens, libraries and principle rooms, we like the elegance that classical order, balance and harmony produces, and as fine cabinet-makers it suits us too. Classical design is steeped in tradition, and we enjoy making furniture in the traditional way, using hardwood that's joined together with mortice and tenons, mason's mitres and halving joints. It's a tried and tested formula.
Around 12 years ago we began to notice a significant shift in furniture and room style, particularly among the super-prime homeowner in London. These buyers typically came from countries such as Russia and India where they mostly hid their wealth. Arrival on the safer streets of London gave these wealthy incomers a new found freedom to become more ostentatious, and overt displays of wealth through interior design became a popular route for those wanting to make a mark on their newly acquired English home.
The consequences were often terrifying. Suddenly clients begun to request furniture made from metal effect spray coated panelling and Swarovski crystal covered shoe shaped baths. Interior design became a heady mixture of luxury hotel interiors crossed with theatre. Design became depressingly temporary.
These bouts of interior design bling one-upmanship became more and more extravagant, many in the quest for elegance. In many cases, it failed horrifically.
More recently however we've noticed a welcome renaissance, with more discerning clients beginning to understand that elegant design is actually best achieved through gentility and restraint. An introduction to the English countryside, its pursuits and architecture has also managed to educate some buyers to the more muted ways of successful English classical furniture and interior design.
Wealthy buyers are now beginning to understand that quality English interior design and architecture is about style, grace, understated beauty, and above all, permanence. They are beginning to realise that it does not pay to be on-trend with interiors. Libraries, kitchens and dressing rooms cannot be replaced every time a new fashion emerges.
Our clients homes are too important to be treated simply as temporary or artificial stage sets with shelf lives, and the furniture design work we undertake here at Artichoke needs to have this air of permanence before it can be presented to the client. For joinery and fitted furniture to truly deliver an air of permanence, it needs to look comfortable and natural in its surroundings.
Restrained elegance is a subtle way of saying so much more
There are many ways of introducing permanence into design, but one sure fire method is to deploy classically inspired design treatments, mouldings, shapes, balance and proportion. When executed well it delivers breathtaking glamour that far outstrips anything that a bronze and crystal adorned flat door panel will ever deliver in its short lifetime.
What wealthy buyers have begun to understand it appears, is that less is more. Or to quote the architect Ludwig Mies van der Rohe, "I don't want to be interesting. I want to be good."
Posted in ArchitectureTagged classical interior design, classical interiors, english architecture, english country house, english interiorsLeave a Comment on A Welcome Return to Classicism
The Start of Formal English Architecture
Posted in Dec 2015 by Andrew Petherick
As a craft based design business focussed on creating bespoke kitchens and furniture to integrate successfully into the fabric of buildings, we need a thorough understating of them, their histories and English architecture in general. We spend a great deal of time in buildings of all types, surveying them, designing their interiors and fixing into their structures.
We spend a significant amount of our time working in period property, so a firm grasp of their architecture, why they are shaped they way they are, what their fabric is and make up is very important to us. In a geeky sort of way we also find it fun.
Dinder House, where we created the bespoke kitchen
The First Architects
During the early 1700s and before, the building trade was actually run primarily by craftsmen; mainly by joiners and carpenters. This makes a great deal of sense; most buildings of the time were timber framed. These craftsmen all had at least seven years of apprenticeships under their belts and were people of considerable skill.
During this period, the term 'architect' was used by anyone who could get away with it. Prior to the introduction of formal Italian inspired classical architecture by Inigo Jones in the mid 1600's, English architecture was essentially medieval in form, with most houses being built organically and of timber frames with no formal design organisation, proportion or layout to them. There was no real need for architects. Most new buildings were designed by builders. There was no such thing as English Architecture in a formal sense.
The introduction of formality in building design came through classical architecture. Classical English architecture's more rigid design requirements meant the need for someone to plan and design buildings more carefully. It also required new materials to achieve the various shapes, mouldings, carvings etc and this meant a greater understanding of the materials capabilities, stresses and loads.
Banqueting House was the first building to be completed in the new neo-classical style.
To begin with, this new role of architectural designer was taken on by the joiner or craftsmen (occasionally it was also taken on by members of the landed gentry, plump with inspiration from their grand tours of Europe).
As you can see from the architecture of Banqueting House, Classical and Georgian architecture is complex; its shapes, mouldings, facades, heirarchys, planes and orders require the use of knowledge, academic formulas and new geometry that wasn't necessary in the construction of timber framed houses. It's introduction to England presented an opportunity for many craftsmen to better themselves, and many stepped up to the plate.
Two things typically happen when a new and game-changing thinking is introduced to any society. Firstly, an influx of quasi-specialists on the new subject appear, and in this case these were the people closest to the coal face; craftsmen and builders. Secondly, there is a publishing revolution to support the thirst for knowledge (the same thing happened during the introduction of the internet on the mid 1990's).
Both happened during the introduction of classical architecture, particularly in the publishing of building pattern books to help educate the uninitiated about classical architecture's forms and functions. Initially, these books were written by craftsmen for fellow craftsmen, and later they were written by new architects for potential clients. These books were used by builders to copy from, and they formed the pattern around which London's early classical architectural structure and then Georgian architecture was born. These books also started formalising the way in which these new building designs were physically erected, and the most famous reference book at the time, The art of drawing and working the ornamental parts of architecture, was written by Batty Langley. Click this link to see some of our favourite plates from the book.
Main Georgian Architectural Materials
A formality of design also led to formality of materials.
Brick – The primary material used to build buildings in Georgian London was brick made from the clay beds of London. The exterior bricks were the best quality, with garden walls, party walls etc often being constructed from 'place' bricks which were as much ash as they were clay and often prone to defects. Knowing the quality of what we're fixing into is important for Artichoke; we often design quite technically complex architectural pieces such as minstrel's galleries or large bespoke kitchens with heavy materials, and we need to know well in advance how robust the structure we're fixing into actually is. There are a number of times I can think of when one of our installation team has attempted to drill into bricks that have simply disintegrated.
Bedford Square was one of London's first Georgian Squares (it was built for the Duke of Bedford) and used London stock bricks.
'Place' bricks were the cheapest, with the most expensive being 'Cutting' bricks, a brick made from fine clay and capable of being very accurately cut. These bricks were usually reserved for exterior decorative architectural detail such as door or window arches.
Timber – The main timber types used by builders of the time were English oak and Baltic fir (Russian Deal is another name for it). English oak was considered a premium timber, as it still is now, and Baltic fir was used for most things structural. It was also used for interior architecture and was regularly painted and gilded. Before Georgian times, Baltic Fir was scraped and left unfinished, only being painted with the introduction of the Georgians. Our finishers need to be comfortable in being able to match 300 year old Baltic Fir; we have had do just this for a recent project in Upper Brook Street in London.
Oak was reserved for the best and most visible structural work such as window frames and some interior architectural detailing. Mahogany bought back from the West Indies was often used on high-end residences for panelling, hand-rails, panelled doors and so on.
Stone – Stone was used less in Georgian architecture than one may think, certainly for many of the more domestic London townhouses. It was usually Portland stone that was sometimes used decoratively in London homes for items such as window dressings, exterior cornices and porches.
For Artichoke, a good understanding of these materials, proportions and the English architecture it makes up is vital, not just because it helps us gain an understanding of what we're fitting our work into, but also because it often informs our design decisions.
We cannot design a bespoke kitchen or library in a country house unless we have a thorough understanding of that house, its architecture and materials. Architecture informs our work, and for us, this is vital, as with the classical builders and architects of the time, our legacy is in our work.
Posted in ArchitectureTagged classical architecture, english architecture, london architecture, period architectureLeave a Comment on The Start of Formal English Architecture
The Flexibility of Formal Architecture
As designers of bespoke kitchens, libraries and architectural joinery for period country houses, we often need to delve into the past to gain inspiration, and often that journey heads towards classical design.
It's a common misunderstanding that formal architecture is rigid and inflexible. In fact we've found the opposite to be the case.
Granted, certain shapes and mouldings are very much guided by geometry, and certain entablatures and orders are to be used in certain specific ways, but the rest seems very much down to the architect. In fact, it's this geometry that's been the reason for classical architecture's success around the world. Geometry gave the architecture a formula that meant it could be copied, time after time, beautifully, in the same way, without risk of changing the proportions and nature of the work. It was, in many ways, architecture by numbers.
One of our favourite reference books for classical interior architecture, bespoke classical kitchens and architectural joinery projects is The city and country builder's and workman's treasury of designs, written in 1750 by Batty Langley.
During the early periods of English Architecture it was not uncommon for architects to be from other trades, such as joiners, cabinet-makers or even, in Langley's case, landscape gardeners. Much of London's early architecture was in-fact "borrowed" from books such as his, and we have referred to many of them ourselves when designing bespoke kitchens or architectural joinery in classical country houses.
These are some of our favourite plates from this period of English architectural publishing.
The geometry of doorways and door architraves
The geometry of pediments and cornice-work
The classical orders used in cornices
We used this plate when working with Craig Hamilton on the kitchen at Williamstrip Park in Gloucestershire. The kitchen included an elliptical niche which we coopered
What is so interesting about these plates are that they show so clearly the reason that classical architecture gained such popularity around the World from its origins in Athens and Rome. The use of geometry provided a known formula that could be copied and exported through books such as Langley's. This allowed the proportions to be kept and thus copied repeatedly in the same beautiful way, time after time.
The best example of the exporting of architecture was the use of Langley's book during the building of George Washington's home Mount Vernon. The builder used Plate 51 from The city and country builder's and workman's treasury of designs when building the Palladian window below.
The original plate from Langley's book
The exact copy from Langley's book, in the same proportions at Mount Vernon
Posted in ArchitectureTagged classical architecture, formal architecture, period architecture, traditional architectureLeave a Comment on The Flexibility of Formal Architecture
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Inactivated and fowlpox virus (FP)-vectored vaccines have been used to control H5 avian influenza (AI) in poultry. In H5 AI endemic countries, breeder flocks are vaccinated and therefore, maternally-derived antibodies (MDA) are transferred to their progeny. Results of three immunogenicity and one efficacy studies performed in birds with or without MDA indicated that the immunogenicity of an inactivated vaccine based on a H5N9 AI isolate (inH5N9) was severely impaired in chicks hatched from inH5N9-vaccinated breeders. This MDA interference was lower when breeders received only one administration of the same vaccine and could be overcome by priming the chicks at day-of-age with a live recombinant FP-vectored vaccine with H5 avian influenza gene insert (FP-AI). The interference of anti-FP MDA was of lower intensity than the interference of anti-AI MDA. The highest interference observed on the prime-boost immunogenicity was in chicks hatched from breeders vaccinated with the same prime-boost scheme. The level of protection against an antigenic variant H5N1 highly pathogenic AI isolate from Indonesia against which the FP-AI or inH5N9 alone was poorly protective could be circumvented by the prime-boost regimen in birds with either FP or AI MDA. Thus, the immunogenicity of vaccines in young chicks with MDA depends on the vaccination scheme and the type of vaccine used in their parent flocks. The heterologous prime-boost in birds with MDA may at least partially overcome MDA interference on inactivated vaccine.
Vaccines are useful tools to control avian influenza (AI) especially when biosecurity and stamping out strategies alone are not successfully implemented -. Inactivated vaccines are the most widely used AI vaccines although viral vectored vaccines based on fowlpox (FP) or Newcastle disease virus (NDV) are also used in some countries -. In endemic countries in which AI vaccination is routinely used in breeders, broilers hatch with maternally-derived antibodies (MDA). A few papers reported the negative effect of MDA on inactivated AI and vector vaccines immunogenicity and efficacy in chickens -.
A FP-vectored AI (FP-AI) vaccine expressing the hemagglutinin (HA) gene from the A/turkey/Ireland/1378/1983 H5N8 highly pathogenic AI (HPAI) isolate has been used in broilers since 1998 in low pathogenic avian influenza (LPAI) H5N2 endemic areas of Central America. One of the main advantages of this FP-AI vaccine is that it is administered to one-day-old chicks by the subcutaneous route at the hatchery whereas inactivated AI vaccines are usually administered in the farms at a later age. The FP-AI vaccine is also a DIVA (differentiating infected from vaccinated animals) vaccine since it induces an immune response against the HA alone and some commercially available enzyme-linked immunosorbent assays (ELISA) are designed to detect the antibody response against the nucleoprotein (NP); these ELISAs can therefore detect infection in a FP-AI vaccinated flock. The FP-AI efficacy in presence of FP and/or H5 AI virus MDA has been shown in a highly pathogenic AI (HPAI) as well as low pathogenic AI (LPAI) Mexican H5N2 challenge model ,. This FP-vectored vaccine was also shown to be efficacious against different HPAI H5N1 isolates in SPF chicks ,. The objective of the studies presented here was to further analyse the effect of MDA on FP-AI vaccine immunogenicity and to evaluate the efficacy of a prime-boost regimen (priming with FP-AI and boosting with inactivated vaccine) in birds with FP or AI MDA.
Three AI vaccines were used: a classical inactivated and two recombinant FP vectored vaccines. The whole virus beta-propiolactone (BPL)-inactivated vaccine (inH5N9) contained the Eurasian isolate A/chicken/Italy/22A/98 [H5N9] (Genbank HA gene: ABR37720), propagated on embryonated SPF eggs (experimental vaccine prepared by Merial Italia S.p.A.). The inactivated antigen (H5N9-It) was formulated in a water-in-oil adjuvant similar to those used in classical avian inactivated vaccines. Two different experimental formulations of H5N9-It inactivated vaccine (inH5N9) were used. For studies 1 and 2, the vaccine was formulated at 0.5 mL (350 HA units) per dose and for studies 3 and 4, it was formulated at 0.3 mL (122 HA units) per dose. The first vectored vaccine contained an experimental FP recombinant (vFP2211) expressing a synthetic hemagglutinin (HA) gene derived from the amino acid sequence of the HA from A/chicken/Indonesia/7/2003 clade 2.1.1 H5N1 HPAI isolate (GenBank: ABO30346) that was modified at the cleavage site (RERRRKK RGLF mutated into RET RGLF) . The other FP vectored vaccine was the TROVAC®-AIV H5 (batches TAH5RF064 (study 2), RD021 (study 3), and RD030 (study 4) produced by Merial Select, Inc., Gainesville, Georgia, USA) vaccine licensed in the USA and other countries. This vaccine contains the FP recombinant vFP89, which expresses the HA gene (Genbank: HMIVT8) of a HPAI H5N8 virus isolated in Ireland (A/turkey/Ireland/1378/83 [H5N8]). The FP vector (TROVAC® vector), insertion site and promoter were the same for the two FP vectored vaccines. It was derived from the seed of the commercial FP vaccine DIFTOSEC CT® (batch 5DCC3511 (breeders of study 1 and 2) and batch 7DCC3801-A (breeders of study 3 and 4) produced by Merial S.A.S., Lyon, France) that was also used as the FP vaccine in our studies.
Five groups of 10 one-day-old chickens were included in this study. These birds were hatched either from unvaccinated SPF hens or from SPF hens vaccinated twice with inH5N9 (0.5 mL) at 3 and 17 weeks of age respectively. These hens were also vaccinated with FP vaccine at 9 weeks of age. Serum was collected from 15 out of 19 hens at 36 and 44 weeks of age for AI serology. The chicks hatched from eggs collected from 42 week-old vaccinated hens had either no MDA or both anti-FP and anti-H5N9 maternal antibodies (named hereafter MDA-FP + H5N9). Chicks without MDA were vaccinated with vFP2211 at 3 log10 TCID50/dose at day 0 (D0). Chicks with MDA-FP + H5N9 were vaccinated either with vFP2211 at 3 log10 TCID50/dose at D0, with vFP2211 at 5 log10 TCID50/dose at D0, with vFP2211 at 5 log10 TCID50/dose at D0 and 0.5 mL of inH5N9 at day 21 (D21) (prime-boost), or with 0.5 mL of inH5N9 at D21 only. The design of this study is shown in Table 1. Vaccination was performed subcutaneously in the nape of the neck at D0 for vFP2211 and intramuscularly in the breast at D21 for inH5N9. Blood was taken 3 weeks after the first vaccination and 3 and 9 weeks after the second vaccination to assess the seroconversion. The level of MDA was evaluated from the serum of 10 additional chicks euthanized after hatch.
Chickens were hatched from unvaccinated (SPF) or vaccinated (MDA) hens. Chicks with H5N9, FP, H5N8 and/or H5N1 MDA were the progeny of hens vaccinated with inH5N9, fowlpox (FP), vFP89 (HA gene from A/turkey/Ireland/1378/83 [H5N8]) and/or vFP2211 (HA gene from A/chicken/Indonesia/7/2003 [H5N1]), respectively. The groups of 9 to 10 one-day-old birds received either vFP2211 or vFP89 or inH5N9 at D0, followed by a boost of inH5N9 at D21 (studies 1and 2) or at D14 (studies 3 and 4). In addition, a HPAI H5N1 challenge was performed in study 4.
*H5(N8) MDA are directed against the HA only of A/turkey/Ireland/1378/83 [H5N8]. Breeders of these chicks were indeed vaccinated with a fowlpox expressing the HA of H5N8 (vFP89).
Three groups of 10 one-day-old chickens were included in this study. One group of SPF birds was immunized with vFP89 at 3 log10 TCID50/dose at D0 and with 0.5 mL of inH5N9 at D21 (prime-boost in SPF chickens without MDA). The birds of the two other groups were hatched from SPF hens vaccinated twice with inH5N9 (0.5 mL) at 3 and 17 weeks of age and once with FP vaccine at 9 weeks of age; serum was collected from 15 out of 19 hens at 60 and 68 weeks of age for AI serology. The chicks from these two other groups were hatched from eggs collected from 62 week-old vaccinated hens and had both anti-FP and anti-H5N9 maternal antibodies (MDA FP + H5N9). One of these 2 other groups was vaccinated with vFP89 at 3 log10 TCID50/dose at D0 and with 0.5 mL of inH5N9 at D21 (prime-boost), and the other at both D0 and D21 with inH5N9 (0.2 and 0.5 mL respectively) (see Table 1). Vaccination was performed subcutaneously in the nape of the neck at D0 and intramuscularly in the breast at D21. Blood was taken 3 weeks after the first vaccination and 1 and 3 weeks after the second vaccination to assess the seroconversion. The level of transmitted MDA was evaluated from the serum of 7 additional chicks euthanized after hatch.
Eight groups of 10 one-day-old chickens were included in this study. Birds were hatched from 4 different groups of hens: (1) non-vaccinated SPF, (2) SPF vaccinated 3 times with inH5N9 (0.3 mL) at 3, 6 and 16 weeks of age, (3) SPF vaccinated twice with FP vaccine at 4 and 12 weeks of age or (4) SPF vaccinated with vFP89 at 3 log10 TCID50/dose at D0, FP vaccine at 12 weeks of age and with 0.3 mL of inH5N9 at 16 weeks of age. Serum was collected from 10 out of 15 hens from group (2) and from 10 out of 20 hens of group (4) at 20 and 28 weeks of age for AI serology. The chicks were hatched from eggs collected from 24 week-old vaccinated hens and had either (1) no MDA, (2) anti-H5N9, (3) anti-FP or (4) anti-FP, anti-H5(N8) (against A/turkey/Ireland/1378/83 [H5N8] HA only) and anti-H5N9 maternal antibodies. These chicks were vaccinated with vFP89 at 3 log10 TCID50/dose at D0 and with 0.3 mL of inH5N9 at day 14 (D14) (prime-boost) or only once at D14 with 0.3 mL of inH5N9 (see Table 1). Vaccination was performed subcutaneously in the nape of the neck at D0 and intramuscularly in the breast at D14. Blood was taken 2 weeks after the first vaccination and 2 and 4 weeks after the second vaccination to assess the seroconversion. The level of MDA was evaluated from the serum of 5 additional chicks euthanized after hatch.
Eight groups of 9-10 one-day-old chickens were included in this study. Birds were hatched from 3 different groups of hens: (1) non-vaccinated SPF, (2) SPF vaccinated twice with FP vaccine at 4 and 12 weeks of age, (3) SPF vaccinated 3 times with inH5N9 at 3, 6 and 16 weeks of age. Serum was collected from 10 out of 15 hens from group (3) at 52 and 60 weeks of age for AI serology. The chicks were hatched from eggs collected from 58 week-old vaccinated hens and had either (1) no MDA, (2) anti-FP or (3) anti-H5N9 maternal antibodies. Chicks without MDA were vaccinated either with vFP89 at 3 log10 TCID50/dose at D0 or remained unvaccinated. Chicks with anti-FP MDA were vaccinated with vFP89 at D0 and with 0.3 mL of inH5N9 at D14 (prime-boost) or at D14 only with 0.3 mL of inH5N9. Chicks with anti-H5N9 MDA were either non-vaccinated, vaccinated with vFP89 at D0, vaccinated with 0.3 mL of inH5N9 at D14, or vaccinated with vFP89 at D0 and with 0.3 mL of inH5N9 at D14 (prime-boost) (see Table 1). Vaccination was performed subcutaneously in the nape of the neck at D0 (vFP89) and intramuscularly (inH5N9) in the breast at D14. All birds were challenged by the intranasal route at day 28 (D28) with 106 EID50 per bird of the Indonesian A/chicken/West Java-Subang/29/2007 HPAI H5N1 isolate classified in 2.1.3.2 subclade . The HA sequence of the A/chicken/West Java/29/002/2007 isolate available in GISAID's EpiFlu™ Database  (Accession number EPI533441) was used for sequence analysis. This isolate was previously used in a published vaccination-challenge study  and is one of the antigenic variants that emerged in Indonesia as early as 2005 . Morbidity and mortality were recorded during 2 weeks after challenge and oropharyngeal swabs were collected 2 and 4 days post-challenge to evaluate viral load by quantitative real time RT-PCR. Blood was taken 2 weeks after the first vaccination (D14) and 2 weeks after the second administration before challenge (D28). The level of MDA was evaluated from the serum of 5 additional chicks euthanized after hatch.
The identity and similarity between the HA1 amino acid sequence of the vaccine strains, the challenge isolate and the viruses used as antigens in the HI test (Table 2) were obtained using the Blastp software of the National Center for Biotechnology Information .
The HA gene of A/turkey/Ireland/1378/83 [H5N8] (tk/Ire/83 H5N8) and of A/chicken/Indonesia/7/2003 [H5N1 clade 2.1.1] (ck/Ind/03 H5N1) is inserted into the genome of fowlpox recombinant vFP89 and vFP2211, respectively. The A/chicken/Italy/22A/98 [H5N9] (ck/Ita/98 H5N9) is used in the inactivated inH5N9 vaccine. The NIBRG14 and NIBRG23 reverse genetics constructs contain the HA and NA genes from A/Vietnam/1194/2004 [H5N1 clade 1] (Vtn/04 H5N1) and from A/turkey/Turkey/1/2005 [H5N1 clade 2.2] (tk/Tur/05 H5N1), respectively. The A/chicken/West Java-Subang/29/2007 [H5N1 clade 2.1.3.2] (ck/WJ/07 H5N1) was used for the challenge in study 4.
The serological analyses were performed using the hemagglutination inhibition (HI) tests. Tests were conducted according to standard procedures, using 4 HA units/well. Different antigens were tested to check for cross-reactivity of the serums towards different recent H5N1 strains. The BPL-inactivated A/chicken/Italy/22A/98 [H5N9] (homologous to the inactivated vaccine strain) was tested on serums from all studies. The BPL-inactivated NIBRG14 H5N1 antigen was used on serums from studies 1 and 2; the NIBRG14 is a 6/8 PR8-based reverse genetics mutant containing the HA (Genbank: ACR48874) and NA of the A/Vietnam/1194/2004 clade 1 H5N1 isolate. The BPL-inactivated NIBRG23 H5N1 antigen was used on serums from study 3; the NIBRG23 is a 6/8 PR8-based reverse genetics mutant containing the HA (Genbank: ABQ58921) and NA of the A/turkey/Turkey/1/2005 clade 2.2 H5N1 HPAI isolate. The H5N8 A/turkey/Ireland/1378/83 antigen, homologous to the HA used in the vFP89 vaccine was also used in studies 2, 3 and 4 and the BPL-inactivated A/chicken/West Java-Subang/29/2007 was used in study 4. The serums of breeders were also tested with the H5N9 antigen as well as with the NIBRG14 (studies 1 and 2) or NIBRG23 (studies 3 and 4) H5N1 antigen. The HI tests on serums from breeder hens were performed at Merial; those from their progeny of studies 1 to 3 and from study 4 were performed at Merial and SEPRL, respectively. The first tested serum dilution was 1 log2 (studies 1 to 3) and 3 log2 (study 4).
The level of viral shedding after challenge was evaluated in study 4 by M-based real time reverse transcriptase (RT) PCR on RNA extracted from swabs as previously described .
Statistical analyses were performed using the STATGRAPHICS and SAS/PC softwares. Analysis were carried out by applying the one-way analysis of variance (1-ANOVA) or two-way analysis of variance (2-ANOVA) with factors "origin of the birds" and "vaccination scheme" and pairwise multiple comparison procedure by the Student's t-test. Statistical difference was based on two-tailed tests of the null hypothesis resulting in p-value of 0.05 or less. A value of 0 (studies 1-3) and 2 log2 (study 4) were set for serums that tested negative for statistical analysis.
Mean H5N1 and H5N9 HI titers in the breeder hens before and after egg harvest and in their one-day-old progeny are presented at Table 3 for the 4 studies. The mean HI titers in the progeny were lower (by 1 to 4 log2) than in their parent hens.
Breeders hens were vaccinated with inH5N9 and fowlpox (H5N9 + FP), inH5N9 only (H5N9) or with vFP89, inH5N9 and fowlpox (H5(N8) + H5N9 + FP). Their serums were taken at two time points flanking the time of harvest of their eggs. Serums of their progeny were sampled at 1 day-of-age. NIBRG14 and NIRG23 H5N1 antigens were used in studies 1 & 2 and studies 3 & 4, respectively.
*mean HI titer ± standard deviation in log2.
Assessment of seroconversion was performed at the time of first (D0) or second (D21) vaccination and after the second vaccination (day 42 (D42) and 84 (D84)). Mean HI titers using two different antigens (H5N1 clade 1 and H5N9) are presented in Figure 1 and the HA1 amino acid homologies between the vaccine and HI antigens are shown in Table 2. The vFP2211 at 3 log10 TCID50/dose induced a low antibody response (2-3 log2) with both antigens that peaked between D21 and D42 in SPF chicks without MDA. In birds with FP/H5N9 MDA, the induced antibody response was decreased by 1-2 log2 suggesting an interference of MDA on vFP2211 immunogenicity. This difference was statistically significant (p = 0.046) with the H5N1 antigen at D21. Increasing the vFP2211 dose to 5 log10 did not increase the HI titers in presence of MDA. One administration of the inH5N9 vaccine in birds with MDA induced an antibody response of 5 and 3 log2 against homologous H5N9 and heterologous H5N1 antigens, respectively. When birds were primed with vFP2211 (dose of 5 log10), the antibody titers after the inH5N9 boost were significantly higher than after the inH5N9 alone. Interestingly, titers against both H5N1 and H5N9 antigens were similar. Overall, results of study 1 indicated a slight interference of FP and/or H5N9 MDA on vFP2211 immunogenicity. However, vFP2211 priming before inH5N9 administration significantly increased the HI titers after the boost indicating an efficient vFP2211 priming despite the presence of MDA.
Study 1 Kinetics of HI antibody titers induced by different vaccines. Mean HI titers (log2) using (A) H5N1 clade 1 (NIBRG14) and (B) H5N9 antigens in the serum of five groups of vaccinated chickens. One group of SPF chickens was vaccinated with 3 log10 of vFP2211. The four other groups had H5N9 and FP MDA and were vaccinated either with 3 or 5 log10 of vFP2211 at D0, with inH5N9 at D21, or with 5 log10 of vFP2211 at D0 followed by inH5N9 at D21 (prime-boost). HI titers were evaluated at D0, D21 (3 weeks after the vaccination with vFP2211), D42 and D84 (3 and 9 weeks, respectively, after the 2nd vaccination with inH5N9 in the prime-boost group).
Assessment of seroconversion induced by a homologous (inH5N9 + inH5N9) or a heterologous (vFP89 + inH5N9) prime-boost scheme was performed at the time of first (D0) or second (D21) vaccination or after the second vaccination (D28 and D42) in SPF chickens or chickens with FP and H5N9 MDAs. Results of the HI test using three different antigens (H5N1 clade 1, H5N8 and H5N9) are presented in Figure 2 and the HA1 amino acid homologies between the vaccine and HI antigens are shown in Table 2.
Study 2 Kinetics of HI antibody titers induced by different vaccines. Mean HI titers (log2) using (A) H5N1 clade 1 (NIBRG14), (B) H5N8 and (C) H5N9 antigens on D0, D21 (3 weeks after the 1st vaccination), D28 and D42 (1 and 3 weeks after the 2nd vaccination, respectively). Two groups of chicks with H5N9 and FP MDA were vaccinated either with vFP89 at D0 followed by inH5N9 at D21 (prime-boost) or inH5N9 at both D0 and D21. The third group was SPF chicks without MDA vaccinated with the prime boost.
After the first inH5N9 vaccine administration at D0, no clear seroconversion was observed in birds with MDA at D21. In SPF chicks, the vFP89 induced at D21 a mean homologous H5N8 HI titer of 4.5 log2. However, no H5N8 seroconversion was observed with this vaccine in birds with MDA indicating that FP and/or AI MDA had negative impact on vFP89 AI immunogenicity.
In presence of anti-FP and anti-H5N9 maternal antibodies, the group vaccinated at both D0 and D21 with inH5N9 had surprisingly no seroconversion against the H5N1 and H5N8 antigens and a very low seroconversion against the H5N9 homologous antigen after the 2nd administration suggesting an interference of the MDA on the inH5N9 immunogenicity. In contrast, the group receiving the vFP89 priming at D0 followed by inH5N9 at D21 clearly showed a seroconversion in presence of MDA for the 3 antigens that was significantly higher at D28 for the H5N8 antigen and at D42 for all 3 antigens than the groups receiving 2 times inH5N9. However, the same prime-boost scheme in SPF chicks induced higher HI titers than in birds with H5N9 + FP MDA. The HI titers in SPF without MDA were significantly higher with H5N8 and H5N9 antigens at both D28 and D42 (Kruskal-Wallis test H5N8 antigen: p < 0.01 and p = 0.04, respectively; H5N9 antigen: p = 0.02 and p = 0.04, respectively) but not with the heterologous H5N1 antigen (Kruskal-Wallis test: p = 0.71 and 0.69, respectively). Note that the identity and similarity between the HA1 amino acid sequence of the vaccines and the H5N1 clade 1 (NIBRG14) HI antigen was lower or equal to 90% and 95%, respectively (Table 2).
Chickens with or without MDA (SPF) were used in this 3rd study. Chicks with MDA had either (1) H5N9 MDA, (2) FP MDA or (3) H5(N8) + H5N9 + FP MDA (see Materials and methods). They were vaccinated with vFP89 at D0 and inH5N9 at D14 or with inH5N9 only (see Table 1). Assessment of seroconversion in the vaccinated birds was performed at D0, D14, D28 and D42. HI test was performed with three different antigens (H5N1 clade 2.2, H5N8 and H5N9). The HA1 amino acid homologies between the vaccines and the H5N1 clade 2.2 (NIBRG23) HI antigen were similar than between the vaccines and the H5N1 clade 1 (NIBRG14) antigen used in studies 1 and 2 (Table 2).
The HI titre kinetics in SPF, H5N9 MDA or H5(N8) + H5N9 + FP MDA birds vaccinated only once with inH5N9 at D14 is shown in Figure 3. A clear interference of AI MDA was observed on the immunogenicity of inH5N9 vaccine and this interference was higher in H5N9 MDA chicks than in H5(N8) + H5N9 + FP MDA chicks, the mean HI titers at D42 being 3-4 log2 below those obtained in SPF birds. Indeed, HI titres in H5N9 MDA chicks at D42 were significantly lower (t-test: p = 0.022 and p < 0.001 for H5N1 and H5N9 antigens, respectively) than those obtained in H5(N8) + H5N9 + FP MDA chicks; at D28, a lower HI titer in H5N9 MDA versus H5(N8) + H5N9 + FP MDA birds was also observed for both H5N8 and H5N9 antigens but the difference was significant (p = 0.003) with the H5N9 antigen only. These results suggest that the level of MDA interference on inH5N9 immunogenicity depends on the number of inH5N9 administrations given to the breeders. In chicks with H5N9 MDA, the vFP89 priming at D0 could clearly overcome the interference of H5N9 MDA on inH5N9 vaccine D14 immunogenicity, since on both D28 and D42 and for the 3 antigens, the HI titres were significantly higher than those induced after one inH5N9 administration to H5N9 MDA chicks (t-test: p = 0.02, p = 0.002 and p = 0.003 at D28, and p = 0.01, p = 0.0001 and p = 0.017 at D42 for H5N1, H5N8 and H5N9 antigens, respectively) and not different (p > 0.05) from those induced after one inH5N9 administration to SPF chicks (see Figure 4).
Study 3 Kinetics of HI antibody titers induced by the inH5N9 vaccine. Mean HI titers (log2) using (A) H5N1 (NIBRG23), (B) H5N8 and (C) H5N9 antigens on D0, D14, D28 and D42 in groups vaccinated with inH5N9 at D14. The birds of these three groups were SPF (no MDA) or with H5N9 MDA or with H5(N8) + H5N9 + FP MDA.
Study 3 Overcoming MDA-interference on inH5N9 vaccine by vFP89 priming. Mean HI titers (log2) using H5N1 (NIBRG23), H5N8 and H5N9 antigens on D28 and D42 (2 and 4 weeks after the inH5N9 vaccination). Birds were either SPF without MDA or had H5N9 MDA and were vaccinated with vFP89 at D0 and inH5N9 at D14 (birds with MDA) or inH5N9 at D14 only (birds with or without MDA).
HI titer kinetics of birds with or without MDA vaccinated with the prime-boost scheme are shown in Figure 5. The highest antibody response against the 3 antigens was observed in SPF chicks without MDA. A clear MDA interference was observed mainly on D28 in chicks with H5(N8) + H5N9 + FP MDA, for which the titres were around 2 log2 lower than those of the groups with other types of MDA (see Figure 5). This interference was less pronounced on D42. The statistical analysis confirmed this difference on D28, HI titres from chicks with H5(N8) + H5N9 + FP MDA being significantly lower than those in SPF chicks for the three antigens (t-test: p = 0.006, p = 0.002 and p = 0.004 for H5N1, H5N8 and H5N9 antigens, respectively), lower than those in chicks with H5N9 MDA with the H5N8 antigen only (t-test: p = 0.028) and lower than those in chicks with FP MDA with the H5N1 and H5N8 antigens (t-test: p = 0.024 and p = 0.008, respectively).
Study 3 Kinetics of HI antibody titers induced by the prime-boost. Mean HI titers (log2) using H5N1 (A), H5N8 (B) and H5N9 (C) antigens on D0, D14, D28 and D42 in groups vaccinated with the prime-boost (vFP89 at D0 and inH5N9 at D14). The birds of these four groups were SPF or with H5N9 MDA or with FP MDA or with H5(N8) + H5N9 + FP MDA.
In chicks with FP MDA, vFP89-induced H5N8 HI titres at D14 were only slightly (non-significant difference of about 0.6 log2) lower than those obtained in SPF chicks (see Figure 5B). In contrast, these H5N8 HI titres induced by vFP89 at D14 were significantly lower (about 3 log2) in chicks with H5N9 or H5(N8) + H5N9 + FP MDA compared to SPF and FP-MDA birds. These data indicate that the anti-HA insert AI MDA had more inhibitory effect on vFP89-induced H5N8 HI titers than the anti-vector FP MDA. After the inH5N9 boost, HI titres were slightly lower than in SPF (1-2 log2 difference significantly different at D42 and for H5N9 antigen only) suggesting a slight interference of the FP MDA on the vFP89 priming efficiency. In chicks with H5N9 MDA, the HI titers induced by the prime-boost regimen were lower than in SPF but the difference was statistically significant (p = 0.021) at D28 and for the H5N9 antigen only.
Eight groups of chicks with FP, with H5N9 or without (SPF) MDA were used in this 4th study (see Materials and methods and the design in Table 1). Results of serology with H5N8 and H5N9 antigens, clinical protection and shedding after challenge are summarized in Table 4. Results of serology with the A/chicken/West Java-Subang/29/2007 [H5N1 clade 2.1.3.2] challenge strain antigen are not shown since all serums were negative (titer < 3 log2) before challenge (D28). The HA1 amino acid homology of the vaccines with theH5N1 clade 2.1.3.2 challenge strain was indeed lower than with the H5N1 clade 1 or 2.2 antigens (Table 2). Furthermore, the HA1 amino acid sequence of this challenge strain had mutations at critical positions adjacent to the receptor binding site: in particular, it had the S133A and A185E mutations responsible for the antigenic drift of the A/chicken/West Java/30/2007 isolate, and one (I151T) of the 4 significant changes responsible for the antigenic drift of the A/chicken/West Java/119/2010 isolate . The number of positive birds (HI titer ≥ 3log2) and the mean HI titer ± standard deviation in 5 birds with H5N9 MDA sacrificed at 1 day of age were 5/5 at 4.2 ± 1.1 log2 and 4/5 at 3.4 ± 0.9 log2 with the H5N8 and the H5N9 antigens, respectively. After challenge with the Indonesian H5N1 antigenic variant HPAI, all 10 SPF and all 9 MDA H5N9 unvaccinated control birds died 2 days post-challenge (dpc) (mean death time (MDT) of 2.0) confirming the severity of the challenge and the absence of H5N9 MDA protection at 28 days of age. At 2 dpc, high levels of oropharyngeal viral excretion were detected for all unvaccinated control birds (7.8 and 7.3 log10 EID50-equivalents in chicks without or with H5N9 MDA, respectively).
Results of serology, protection and oropharyngeal viral shedding in chicks with or without FP or H5N9 MDA vaccinated or not with vFP89 and/or inH5N9 and challenged at D28 with an HPAI H5N1 Indonesian isolate (A/chicken/West Java-Subang/29/2007).
aNumber of positive birds (HI titer ≥ 3 log2)/total; the mean HI titer ± standard deviation is given between brackets.
bNumber of birds protected against mortality/total; the mean death time (MDT) is given between brackets.
cNumber of positive birds (≥3.8 log10 EID50-equivalent)/total; the mean viral RNA load ± standard deviation is given in log10 EID50-equivalent between brackets; Dpc, day post-challenge.
The vFP89 alone induced homologous H5N8 HI titers at D14 (3.7 log2 mean HI titer) and D28 (5.5log2) in all 10 SPF birds of group 2. In birds with H5N9 MDA, H5N8 seroconversion after vFP89 vaccination was observed in none (0/9) of the birds at D14 (group 7) and in only 5/9 birds (2.9 log2) at D28 (group 6) indicating an interference of H5N9 MDA on vFP89 immunogenicity. In birds with FP MDA, 2/9 birds had detectable H5N8 antibodies (2.3 log2) 14 days after vFP89 vaccination. This decrease of vFP89 immunogenicity in FP MDA birds compared to SPF birds was higher than in study 3 (1.4 log2 and 0.6 log2 decrease in study 4 and 3, respectively). Only one out of ten SPF birds without MDA was clinically protected after the vFP89 vaccine alone (MDT 3.4), whereas none of the 9 vFP89-vaccinated birds with H5N9 MDA survived after challenge (MDT 2.7); this difference was not significant. Viral shedding was detected in all vFP89-vaccinated challenged birds at similar levels as non-vaccinated control (7.1 and 7.7 log10 at 2 and 4 dpc, respectively) in birds with H5N9 MDA (group 6) and at slightly lower levels (6.3 and 6.6 log10 at 2 and 4 dpc, respectively) in SPF birds (group 2). These results indicated a low level of protection induced by vFP89 alone against this variant Indonesian HPAI H5N1 isolate which may further be decreased by the presence of AI MDA.
The inH5N9 alone induced higher homologous H5N9 HI titers and in a higher proportion of birds (mean titer of 5.8 log2; 9 positive/9 birds) at D28 in FP (group 4) compared to H5N9 MDA (group 8) chickens (mean titer of 3.7 log2; 6 positive/9 birds) confirming the negative impact of H5N9 MDA on the inH5N9 immunogenicity. After challenge, 5/9 or 4/9 chickens with FP (group 4) or H5N9 MDA (group 8), respectively, were clinically protected. The mean death time was higher in group 4 (7.3) than in group 8 (5.0). There was no relation between D28 HI titers and clinical protection for group 4 (inH5N9 at D14 in FP MDA) since dead birds pre-challenge H5N8 and H5N9 HI titers (4-6 and 6 log2, respectively) were similar than those in protected birds (4-9 and 5-7 log2, respectively). Interestingly, such relation existed for group 8 (inH5N9 at D14 in H5N9 MDA): all 5 dead birds were negative (<3 log2) with H5N8 antigen, and negative (3 birds) or at minimum titer (3 log2 in 2 birds) with homologous H5N9 antigen whereas all protected birds had detectable D28 H5N8 and H5N9 HI titers ranging from 4 to 6 log2. The viral shedding at 2 dpc of groups 4 and 8 was comparable (about 5 log10 in all but 1 or 2 birds) and 2.9 or 2.3 log10 lower than the unvaccinated controls in FP or H5N9 MDA chickens, respectively. The shedding at 4 dpc was slightly lower in group 4 (mean titer of 4.6 log10 detectable in 4/9 birds) than in group 8 (mean titer of 5.3 log10 detectable in 7/8 birds). No relation was found between D28 HI titers and the level of shedding. These results indicate that this experimental inH5N9 vaccine was only partially protective against this Indonesian HPAI H5N1 challenge and that H5N9 MDA decreased its immunogenicity.
The groups that received the heterologous prime-boost regimen in presence of FP (group 3) or H5N9 (group 7) MDA induced higher HI titers (7.6 vs 5.8 in FP MDA birds and 4.3 vs 3.7 log2 in H5N9 MDA birds) and/or in a higher number of birds (9/9 vs 6/9 in H5N9 MDA birds) than birds that received the inH5N9 only (groups 4 and 8). These birds of group 3 and 7 were also better clinically protected (9/9 and 7/9 (MDT 7.0), respectively) than those of groups 4 and 8 (5/9 (MDT 7.3) and 4/9 (MDT 5.0), respectively). The levels of viral shedding of groups 3 and 7 were comparable to those of groups 4 and 8, respectively. There was no relation between HI titers at D42 and clinical protection or shedding level in birds from groups 3 and 7. The prime-boost regimen improved the immunogenicity and protection levels compared to one inH5N9 administration for all tested condition (anti-vector FP MDA and anti-H5N9 MDA).
Most published studies on the efficacy of avian influenza vaccines in chickens are performed in SPF chickens without MDA. In endemic countries in which vaccination is practiced, breeder flocks are heavily vaccinated due to their very high economic value in both egg and poultry meat productions. Consequently, breeder hens transfer high MDA levels to their progeny which may protect the hatched chicks but may also interfere with vaccine efficacy. Therefore, the level of immunogenicity and protection induced by vaccination in the presence of MDA is important to evaluate. The study of MDA interference is complex since many factors can influence the data including (1) the vaccination protocol and the types of vaccines used in the breeders, (2) the time between immunization of breeder hens and collection of their eggs, (3) the type of birds and in particular their growth rate which influences the kinetics of MDA decrease after hatching, (4) the heterogeneity of MDA levels in the progeny, (5) the age of vaccination and the type of vaccine used in the progeny, and (6) the timing of challenge with a possible protective effect of MDA. Using vector vaccines, both MDA against the vector and against the targeted disease agent need to be evaluated since both may potentially interfere with immunogenicity. Our objective was to evaluate the MDA interference on FP-vectored and inactivated H5 vaccines' immunogenicity and protection. Different vaccination schemes were tested and compared in SPF chicks and chicks with MDA.
Mean HI titers in 1-day-old chicks were lower (by 1 to 4 log2) than those in their parent hens. A similar difference (2.1-2.5 log2) in homologous H5N2 HI titers between the serum of vaccinated broiler breeder hens and their progeny was previously reported ,. The difference is probably due to a low level (about 10%) of embryo IgY absorption from the egg yolk . The low titer in 1-day-old chick may also be due to the incomplete resorption of the yolk sac in the newly hatched chicks. Antibody continues to be absorbed from the yolk sac after hatching  and total serum IgY levels were shown to increase to their maximum value at about 2 days post-hatch . We have observed similar and even higher mean HI titers (up to 1.7 log2 higher) between day of hatch and one week-of-age in SPF white Leghorn type of birds hatched from vaccinated hens (Bublot et al., unpublished results). The small number of birds used to evaluate the mean HI titers in hens and one-day-old chicks may also explain the variable difference between these hen and progeny titers in different studies. Measuring the antibody titer after a couple days of live and in a higher number of animals could give a more accurate level of MDA.
Results of study 1 indicated that FP and/or H5N9 MDA can slightly interfere with vFP2211 immunogenicity. In addition, increasing the dose of vFP2211 by 2 log10 did not induce an increase in HI titers. The immunogenicity of the inH5N9 alone in birds with FP and H5N9 MDA was lower than expected in both studies 1 and 2 but there was no control group of SPF birds receiving the inH5N9 alone to show that this lower immunogenicity was due to MDA interference. However, vFP2211 priming before inH5N9 administration significantly increased the HI titers after the boost in study 1 indicating an efficient vFP2211 priming despite the presence of FP and H5N9 MDA. Interestingly, the HI titers against both H5N9 and H5N1 antigens were similar after the prime-boost. In study 2, the heterologous prime-boost vaccination regimen using the vFP89 + inH5N9 combination clearly induced a higher HI antibody response than the homologous inH5N9 + inH5N9 prime-boost in birds with MDA confirming the superior immunogenicity of the heterologous prime-boost compared to two administrations of inH5N9 vaccine in birds with MDA. However, the immune response induced by the heterologous vFP89 + inH5N9 prime-boost in birds with FP and H5N9 MDA was lower than in SPF birds indicating that MDA interfered on this vaccination scheme.
In the first two studies, chicks with both AI and FP MDA were used and therefore it was not possible to determine if the interference observed was due to the anti-vector and/or to the anti-AI maternal immunity. In addition, the MDA interference on the inH5N9 immunogenicity was not assessed. Studies 3 and 4 were therefore designed to answer to these questions in terms of immunogenicity (both studies 3 and 4) and efficacy (study 4). In study 3, the highest MDA interference (3-4 log2 lower than in SPF at D28 and D42) was observed on the immunogenicity of the inH5N9 vaccine given at 14 day-of age to birds hatched from breeders vaccinated three times with the same inH5N9 vaccine. Interestingly, in birds hatched from breeders vaccinated with a prime-boost regimen including only one administration of inH5N9, the interference was lower (see Figure 3), indicating that the number of vaccination applied to the breeders may influence the immunogenicity of the same vaccine given to the progeny. These results supported the hypothesis that the poor immunogenicity of the inH5N9 vaccine observed in studies 1 and 2 was due to MDA interference. A similar but less important (2 log2) interference of inH5N9 MDA on the inH5N9 immunogenicity was observed in study 4 (groups 4 and 8 in Table 4); this lower interference was probably due to the lower H5N9 MDA titers in chicks from study 4 (Table 3) because the timing of harvest of eggs from breeders was much later in study 4 (58-week-old breeders) than in study 3 (24-week-old breeders). The H5N1 HPAI protection induced by one-shot (D14) of inH5N9 vaccine in birds with or without H5N9 MDA was not significantly different in the tested conditions of study 4. However, birds with H5N9 MDA (group 8) tended to be less protected than those without H5N9 MDA (group 4): (1) one additional bird died (5/9 vs 4/9), (2) mean time to death was 2.3 days shorter (5.0 vs 7.3) and (3) the number of birds shedding the virus at 2 and 4 dpc was higher (total of 15/17 vs 11/18) (Table 4). Interestingly, there was a clear relation between H5N8 and H5N9 HI titers at D28 and clinical protection in birds with H5N9 MDA (all birds with a H5N8 and H5N9 titer ≥ 4 log2 were protected and those with a titer ≤ 3 log2 died) but not in birds with FP MDA (dead birds had similar H5N8 and H5N9 titers (4-6 log2) than protected ones (4-9 log2)). Prediction of protection based on H5N9 and H5N8 HI titers before this Indonesian antigenic variant challenge was therefore valid in birds with but not in birds without H5N9 MDA. At similar inH5N9-induced H5N9 and H5N8 HI titers (4-6 log2) before challenge, all 4 birds hatched with H5N9 MDA were protected whereas only 4/8 birds with no AI MDA resisted the challenge. Additional studies need to be performed to confirm these results and to identify the immune response differences that lead to the same HI titers but a different level of protection in birds with or without AI MDA.
Passive antibody interference on AI inactivated vaccines immunogenicity and efficacy has been reported by different teams. The MDA impact on inactivated vaccine immunogenicity was a drop in HI titers ranging from 2 log2  to up to 8 log2 . Protection levels were however only slightly reduced in birds with MDA after H5N1 HPAI challenge . Abdelwhab et al.  showed that the MDA interference on immunogenicity was higher when the same vaccine was used in the breeders and in the progeny, and when the breeder vaccine antigen was used in the HI test. Our data showing that the MDA interference level was dependent on the breeder vaccination program are in line with these findings. The MDA interference on inactivated vaccine immunogenicity was also strongly suspected in Indonesian broilers when the same vaccine was used in breeders and their progeny . Kim et al.  and Forrest et al.  simulated the presence of MDA by passive transfer of hyperimmune serum in young chicks and showed that such transfer also interfered on the immunogenicity and efficacy of commercial inactivated vaccines. Altogether, these data confirm that MDA transferred from the vaccinated breeders to their progeny interfere with the immunogenicity and efficacy of inactivated vaccine given at least in the first 2 weeks of age, especially when multiple administrations of the vaccine are used in breeders and the same vaccine is used in the progeny. The interference is detected even with low MDA titers and is lower when heterologous inactivated vaccines are used in the progeny. It is therefore important to take into account this interference when designing the vaccination program of breeders and their progeny.
The anti-FP and anti-AI MDA interference on vFP89 immunogenicity could be evaluated only before the boost (D14) in studies 3 and 4. Anti-FP MDA had only a slight negative effect on vFP89-induced H5N8 HI titers in study 3, but in study 4, the negative effect of anti-FP MDA was higher. After the inH5N9 boost in vFP89-primed birds with FP MDA in study 3, the HI titers did not reach the levels obtained after prime-boost immunization of SPF birds (Figure 5). These results suggest that the vFP89 priming efficiency may have been partially inhibited by the FP MDA. The priming effect of vFP89 on inH5N9-induced immunogenicity and protection (study 4; see Table 4) in birds with FP MDA was obvious since the mean HI titer at D28 and the number of protected birds were higher with vFP89 priming (group 3) than in the group without vFP89 priming (group 4). In previous studies performed with vFP89  and with another FP recombinant expressing Newcastle disease virus (NDV) protective genes , no interference of FP MDA could be observed on AI and ND protection. Altogether, these data indicate that there may be a slight negative interference of anti-FP MDA on the immunogenicity of FP vectors but the anti-vector MDA effect on protection seems minor. This low interference of passive anti-FP immunity contrasts with the strong one observed in birds with active FP immunity. Both Swayne et al.  and Iritani et al.  have shown in an AI and a NDV model, respectively, that FP vaccination performed before administration of the FP recombinants severely decreased the induced protection. The low interference of passive FP immunity could be due to the poor level of FP-induced neutralizing antibodies , and the high interference of active FP immunity to the high FP-induced cellular immunity .
The interference of anti-AI MDA on vFP89-immunogenicity in both study 3 and study 4 was more obvious than that of anti-FP MDA. A lower number of seropositive birds and a lower mean HI titer (by approximately 2.5 log2) were detected compared to SPF. These data suggest that interference of MDA directed against both FP and AI observed in studies 1 and 2 on vFP2211 and vFP89 immunogenicity may rather be due to anti-AI MDA than to anti-FP MDA, without excluding the impact of anti-FP MDA. No conclusion can be drawn on the effect of H5N9 MDA on vFP89 efficacy since in the conditions tested in study 4, the vFP89 alone protected only 1/10 SPF birds and 0/9 birds with H5N9 MDA. The poor performance of vFP89 in SPF birds against this Indonesian H5N1 HPAI was surprising since several efficacy studies (,,, M. Bublot, D.E. Swayne and T. van den Berg, unpublished data) performed previously in SPF chickens with other H5N1 HPAI (clade 0, 1, 2.2 and 2.5) showed efficacy levels ranging from 75-100%. The low vFP89 efficacy level in SPF observed here is likely due to the antigenic drift of this Indonesian isolate that contains critical mutations adjacent to the receptor binding site of its HA1 that were shown to be responsible for antigenic drift in Indonesia . A similar poor level of protection induced by inactivated vaccines containing seed strains A/turkey/Wisconsin/1968 (H5N9), A/chicken/Mexico/28159-232/1994 (H5N2), A/turkey/England/N28/1973 (H5N2), A/chicken/Legok/2003 (H5N1), reverse genetic A/goose/Guangdong/1/96 (H5N1), or reverse genetic A/chicken/Vietnam/C57/2004 (H5N3) against another Indonesian antigenic variant (A/chicken/West Java/PWT-WIJ/2006 (H5N1 clade 2.1.3)) has been observed (D.E. Swayne et al., manuscript in review). In previous studies performed with vFP89 in broilers with both AI and FP MDA, there was no significant interference of the MDA on the protection induced against a HPAI H5N2 Mexican challenge model . However, Faulkner et al.  showed recently the interference of passively transferred antibodies on the immunogenicity and H5N1 HPAI protection induced by fowlpox- or NDV-vectored AI vaccines indicating that the level of interference likely depends on the tested conditions. A slight interference of MDA directed against the foreign gene product expressed by the vector vaccine has also been reported for FP-NDV (vFP-ND) by Taylor et al. .
As in studies 1 and 2, the heterologous prime-boost regimen induced higher HI titers in chicks with AI MDA than the inactivated vaccine alone. The vFP89 priming was efficient in birds with H5N9 MDA and override the negative effects of MDA on inH5N9 immunogenicity (Figure 4). In study 4, the protection of the prime-boost in H5N9 MDA was higher (7/9 protected; MDT 7.0) than that of inH5N9 in FP MDA (5/9 protected; MDT 7.3) or in H5N9 MDA (4/9 protected; MDT 5.0) confirming the positive effect of the vFP89 priming in both type of birds. Such priming effect of vectored vaccines has also been observed in a passive antibody transfer study mimicking MDA .
Among the different types of MDA tested (FP, H5N9 or H5(N8) + H5N9 + FP), the highest interference on the prime-boost immunogenicity (Figure 5) was observed at D28 in birds hatched from breeders vaccinated with the prime-boost regimen (H5(N8) + H5N9 + FP MDA) suggesting that, as for inactivated vaccine, interference is higher when the same vaccines and vaccination schemes are used in breeders and their progeny. Several mechanisms responsible for MDA interference have been proposed including (1) antigen removal by elimination of immune complexes, (2) neutralization of live vaccine virus, (3) epitope masking and (4) inhibiting of B-cells by cross-linking of B-cell receptor, antigen, maternal antibodies and the Fcγ receptor IIB. Kim et al.  found that it was the latter mechanism which was mainly responsible for interference in a measles vaccination model in cotton rats. Further studies are needed to better understand the mechanisms of MDA interference in chickens.
The data presented here and recent published work show that AI MDA can potentially interfere on both inactivated whole avian influenza virus and live fowl poxvirus vectored vaccines. Interestingly, the heterologous prime-boost gave the best immune response and the best level of protection. Similar fowlpox vector/inactivated vaccine prime-boost was shown to be highly immunogenic in ducks . There are at least 3 explanations for the highest performance of such heterologous prime-boost ,. Firstly, the two types of vaccine induce different type of immunity: the live fowlpox vector vaccine induces mainly cellular immunity and the inactivated vaccine induced mainly humoral immunity, the combination of two providing a more balanced Th1/Th2 and a broader immune response. Secondly, the fowlpox vector is expressing only HA, the primary protective antigen of influenza virus. After the boost with the whole virus inactivated vaccine, the higher secondary immune response will be directed against the protective HA antigen only and a lower primary immune response will be induced against the other antigens. The broader immunogenicity of a prime-boost regimen using a vector expressing the protective antigen and protein (or another vector) has been shown for different antigens  including recently for influenza in mice, ferrets and monkeys . Thirdly, the HA inserted in the fowlpox vector (from an H5N8 isolate) is different from the HA present in the inactivated vaccine (from an H5N9 isolate). The secondary immune response after the inactivated vaccine boost will be directed against the epitopes common to the two HA antigens. These common epitopes (including epitopes from the HA stem) are thought to be more conserved than the strain specific epitopes resulting in a broader immune response . Heterologous prime-boost regimens with vaccines containing different H5N1 antigens have been shown to provide broader immunity than homologous prime-boost ones with the same vaccine -. Such successive immunization with HA of different origins has also recently allowed to generate broadly reactive H3 monoclonal antibodies  and are used to induce broadly reactive antibodies recognizing the HA stalk . Similar benefits of a prime-boost vaccination regimen in birds with MDA were recently shown in an HPAI H5N1 model with a herpesvirus of turkey vector priming .
Countries such as Indonesia and Egypt in which H5N1 HPAI infection became endemic have not eradicated the infection by vaccination. This failure has many explanations ,, including the suboptimal use of vaccines that seems to have led to the emergence of new antigenic variants against which the classical vaccines are not fully protective ,,. MDA interference on vaccine efficacy likely contributes also to the lack of control by vaccination ,. Results presented here confirm the high MDA interference on inactivated vaccines and suggest that the use of a prime-boost strategy using a fowlpox vector to prime may be able to overcome at least partially MDA interference. Furthermore, the vaccines and vaccination protocol used in breeders and in their progeny need to be different to minimize MDA interference. Additional vaccination/challenge studies need to be done in field conditions to find the optimal vaccination schemes providing the best protection in breeders and in their progeny.
All authors have made substantial contributions to study design, analysis and interpretation of data. ARM, SG and DES have conducted the studies. MB did the sequence comparison analysis. ARM and MB have written the manuscript which was then revised by DES and FXL. All authors read and approved the final manuscript.
This work was supported by the French Ministry of Industry via LyonBioPole (Lyon, France). We thank Sandrine Chanavat-Bizzini, Michelle Duboeuf, J.-F. Piriou, J.-L. Dumas, F. Vermorel, J. Beck for technical assistance and V. Cozette for statistical analysis of study 3. The NIBRG14 and NIBRG23 H5N1 viruses were kindly provided by NIBSC. We thank the Indonesian Ministry of Agriculture for cooperation and providing viruses and sequence data to the veterinary health research and diagnostics organization and laboratories. We gratefully acknowledge the authors, originating (Dr. Teguh Y. Prajitno) and submitting (Koel, B.F.; van der Vliet, S.; Burke, D.F.; Bestebroer, T.M.; Bharoto, E.E.; Herliana, I.; Laksono, B.M.; Xu, K.; Skepner, E.; Russell, C.A.; Rimmelzwaan, G.F.; Perez, D.R.; Smith, D.J.; Prajitno, T.Y.; Yasa, I.W.W.; Osterhaus, A.D.M.E.; Fouchier, R.A.M.) laboratories of the Indonesian H5N1 challenge HA sequence from GISAID's EpiFlu™ Database, on which this research is based; all submitters of data may be contacted directly via the GISAID website . TROVAC and DIFTOSEC are registered trademarks of Merial in the United States of America and in France, respectively, and elsewhere. BLAST is a registered trademark of the National Library of Medicine.
ARM, SG, FXLG and MB are employees of MERIAL, the animal health division of Sanofi company, which is selling avian influenza vaccines. DES declares that he has no competing interests.