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The Round House
The Round House won the National Book Award for fiction.
One of the most revered novelists of our time—a brilliant chronicler of Native-American life—Louise Erdrich returns to the territory of her bestselling, Pulitzer Prize finalist The Plague of Doves with The Round House, transporting readers to the Ojibwe reservation in North Dakota. It is an exquisitely told story of a boy on the cusp of manhood who seeks justice and understanding in the wake of a terrible crime that upends and forever transforms his family.
Riveting and suspenseful, arguably the most accessible novel to date from the creator of Love Medicine, The Beet Queen, and The Bingo Palace, Erdrich's The Round House is a page-turning masterpiece of literary fiction—at once a powerful coming-of-age story, a mystery, and a tender, moving novel of family, history, and culture.
Product Details
|HarperCollins Publishers
|Publication date:
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|Product dimensions:
|5.30(w) x 7.90(h) x 0.90(d)
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About the Author
Louise Erdrich is the author of fifteen novels as well as volumes of poetry, children's books, short stories, and a memoir of early motherhood. Her novel The Round House won the National Book Award for Fiction. The Plague of Doves won the Anisfield-Wolf Book Award and was a finalist for the Pulitzer Prize, and her debut novel, Love Medicine, was the winner of the National Book Critics Circle Award. Erdrich has received the Library of Congress Prize in American Fiction, the prestigious PEN/Saul Bellow Award for Achievement in American Fiction, and the Dayton Literary Peace Prize. She lives in Minnesota with her daughters and is the owner of Birchbark Books, a small independent bookstore.
Hometown:Minneapolis, Minnesota
Date of Birth:June 7, 1954
Place of Birth:Little Falls, Minnesota
Education:B.A., Dartmouth College, 1976; M.A., Johns Hopkins University, 1979
What People are Saying About This
"The novel showcases her [Erdrich's] extraordinary ability to delineate the ties of love, resentment, need, duty and sympathy that bind families together…[a] powerful novel worth reading."
"THE ROUND HOUSE is a stunning piece of architecture. It is carefully, lovingly, disarmingly constructed. Even the digressions demand strict attention."
"Emotionally compelling…Joe is an incredibly endearing narrator, full of urgency and radiant candor…the story he tells transforms a sad, isolated crime into a revelation about how maturity alters our relationship with our parents, delivering us into new kinds of love and pain."
"Erdrich threads a gripping mystery and multilayered portrait of a community through a deeply affecting coming-of-age novel."
"A stunning and devastating tale of hate crimes and vengeance…Erdrich covers a vast spectrum of history, cruel loss, and bracing realizations. A preeminent tale in an essential American saga."
"…a powerful human story…By boring deeply into one person's darkest episode, Erdrich hits the bedrock truth about a whole community."
Customer Reviews
Average Review
Most Helpful Customer Reviews
The Round House by Louise Erdrich just won (like last week!) the National Book Award for fiction. I had this book on my to-read list for a while, but once I heard it won a large award, I had to see what the fuss was about. The Round House takes place in 1988 on an Native American reservation in North Dakota. The reservation is shaken when Joe's mother is brutally raped. Joe and his father, a reservation judge, work to figure out who committed the crime in order to prosecute. This is a good distraction for them, since Mom has completely checked out and spends all her time in her room with the blinds closed. However, solving this crime is not as easy as it seems, because the story is a little more complicated. Joe's only thirteen, but while he and his best friends work through their teenage trials and tribulations, they also work to find answers that they will be able to live with. The Round House was an amazing read. The story was moving, honest, and emotional. You were able to peek into Native American traditions, as well as some of their struggles with the U.S. government. This is definitely a book I recommend! Thank you to Goodreads First Reads and Harper Collins for the copy of this book! Thanks for reading, Rebecca @ Love at First Book
If you haven't read her, this is both a good introduction to her and a bit bewildering. Her writing is amazing - the first sentence will grab you. But some of the references to American Indian history, law and folklore may sail over your head. Also, as before, there are characters re -appearing from earlier novels. Also, if you don't read the New Yorker, you won't necessarily realize that some of the embedded "stories" were in fact short stories previously published. All of that being said - READ IT. She is an amazing writer. She narrates stories that all Americans should know about. And then go back and start with "Love Medicine," her first and go forward. You will relish every minute!
Outstanding, muli~layered and a very good story.
Intricate, enjoyable, and entertaining story, though dealing with social issues that are uncomfortably challenging. Issues are not Anglo-European nor Native American, but human issues. Not all loose ends are tied, but that is how it should be. - Doctor Blue
I'm new to Louise Erdrich, so I can't compare this work with any of her others, but I enjoyed it very much. The protagonist, a 13 year old boy named Joe, is wise beyond his years, and is an unforgettable character. I love the way Erdrich writes from her Native American perspective, giving the reader greater insight and understanding of that culture, while at the same time writing a very good story that is both universal and timeless. The story is complex and disturbing in a way real life stories can be, and I appreciated the fact that the ending to this book left me asking questions instead of tying everything up neatly. I should also say that I found the writing to be exceptional, and I will definitely start reading her other books.
On a reservation in North Dakota, a woman is brutally raped and beaten and nearly killed, but escapes her attacker. What follows throughout this story is a sorting-out, a coming-to-terms, and a desire for justice. The story is narrated by Joe Coutts, a courageous thirteen-year-old Ojibwe boy living on the reservation. His family is "wealthy" by reservation standards, with a nice, but modest, home and plenty to eat. His father Antone is a tribal judge, and his mother Geraldine is something of a tribal genealogist-- it's her job to keep track of family lines and name changes and the like. Geraldine is the woman raped at the beginning of the story, and her son Joe must deal with the feelings this rouses in him, and must attempt, along with his father, to repair their fractured family. The extended family consists of Geraldine's sister Clemence, who lives nearby with their father Mooshum (Joe's grandfather), and her husband Edward. Also Geraldine and Clemence's brother Whitey lives on the reservation, and along with ex-stripper Sonja he runs a gas station on the reservation. Young Joe has the emotional support of his group of friends. Cappy Lafournais is his loyal best friend, and like a brother to him. Zack and cousin Angus round out the group (Angus lives in abject poverty on the res, and it isn't uncommon to see him sporting a black eye or bruised cheek.) This story shines a spotlight on the inability to prosecute many crimes committed against Native Americans, due to the convoluted maze of laws in regard to Natives, reservation grounds vs. non-reservation property, and who is even considered to be Native American (which has turned into a complicated formula of what percent you are this or that.) One thing I had difficulty with at times is the author's writing style. At times it is very clipped and staccato, which is always a bit of a put-off for me. And the dialogue doesn't use quotations, which I always find a bit confusing, as it makes it difficult for me to discern dialogue from thought from narrative. But she definitely has a way with words, and at times I felt my mind say, "Oh!" at the way she expressed something. Overall I found it to be a powerful story, original and unembellished. My final word: Part mystery and part family drama, it's a tragic story, rife with poverty, abuse, alcoholism, death. But overshadowing it all is a sense of hope, of a people who hold a fragile grasp on all of the good that life has to offer, who suck the marrow from life. There is hope in this young boy Joe.
I don't usually identify with stories about preteen boys, but this is story for anyone with a heart or conscience. Don't skip the Afterword. Knowing that this a story that seems to have no end, makes it that much more poignant. It is so well written that the characters will stay in your mind as if you are remembering them as real, not invented. This is a novel but will stay with you as if you lived it.
I found this to be a fantastic novel! However, one thing to note is that there are times where things of a sexual nature are discussed. There is, of course, the rape itself. But there are also events and comments that are very sexual in nature. I found all of this to be in good taste and it didn't disrupt from the important themes that Erdrich has placed in this novel. If the reader is not bothered by sexual themes then there should be no problem with reading this. Like all of Erdrich's work, The Round House makes wonderful use of many literary components; things such as symbolism and motif are used beautifully and contribute greatly to the story. That being said, the reader is highly encouraged to keep a sharp eye on recurring themes and ideas because they truly make the story far more exciting. But of course the novel is great even if certain other literary aspects are overlooked! Another fantastic aspect of this novel is one of the things that Erdrich does best: give insight into what Native American life is like. It is a part of this country that many people overlook and this is an intriguing way to become more informed. A lot of people who have reviewed this novel have been calling it "stupid" and saying that it jumped around. But the literary components really are a huge part of this novel and I feel that perhaps these readers may have overlooked this. As for the "jumping around" comments, it adds to the effect that the novel gives. It also gets the reader to really think about what's going on and get more involved in the way the story is moving. Additionally, people have been discussing their disapproval of the lack of quotation marks. Please, do not let this put you off. I also found this extremely annoying when I began reading this novel but I got used to it once I really got into the story. You must keep in mind, the writer does everything for a reason, including leaving out her quotation marks. This, too, adds to the literary effect. It also gives the reader something to ponder; to question its significance. This novel truly is fantastic, and Louise Erdrich is a fascinating writer. The moods and concepts that she depicts are beautifully conveyed in her detailed imagery and carefully crafted words. I would definitely read this novel again, and I would of course suggest that others read it as well.
Discovering a new, powerful voice, this was my first read of Ms. Erdrich's work. I was instantly struck by her ease in conveying the setting and the characters on the Ojibwe Reservation. Having spent many years with Native folks from several different Nations in our family business, her writing brought me right back into those memories with an astounding accuracy. She also has created some of the most accurate, moving portraits of teen age boys that I have ever read. Putting thoughtful, good people into the heart of impossible evil and telling the story of their journey to re-discover themselves and some sense of justice, brought this reader serious lessons, but not devoid of humor, high spirits or joy along the way. She captures the spirit that sustains Native people caught between cultural pressures and helps clarify to the rest of America, how far we still need to come to provide equal protection under the law. She has also revealed a great deal of her own heart in the process. She is a first rate storyteller not to be missed. Once I read a bit about the author, I realized that there was another spirit alive in her work. It seemed to me to be the same spirit that had a group of traditional Lakota women find the courage to occupy Wounded Knee back in the early 1970s to protest what passed for justice in Pine Ridge. Ms. Erdrich's story reminded me of the power, humor and strength in another Ojibwe from Turtle Mountain. Leonard Peltier
A very moveing glimpse into a little known part of American society.
Living in North Dakota and knowing of the Native American culture in the area, I wanted to read this book. It didn't give me the insight I thought it would into the culture but instead I found an easy book to read that kept me wanting to know how it ended. The author paints a clear picture of her characters and the book could have been longer to give the characters more depth. I first heard of the book as it was highlighted in the Whole Living Magazine and then I heard about it on NPR, a found it to be a sign that I should read it, I am glad that I did.
Great plot...and of course her unique way of delivery.
Good story, and it held my interest. Only complaint is that quotation marks were not used for dialogue, which could be VERY confusing at times The story is told in first-person, so if the dialogue was in first-person too, it was difficult to know whether it was another character speaking, or just the main character narrating.
This novel was a fantastic read. Great writing, amazingly "real" characters, wonderful story. I couldn't put it down. I HIGHLY recommend it to everyone!!!
I had reservations (no pun intended) about this book, but I am so glad I read it. I thought it was worth my time, and that's high praise for any book.
I had a hard time putting this one down. I appreciated the tidbits of history behind the characters, the native humor, difficulties of jurisdiction and tribal sovereignty. This helped fill out the story for all whether or not they have an understanding of life on a reservation. For me, the characters matched someone I knew from my childhood. The story took me through a whirlwind of emotions, from near tears to laughing out loud, and left me satisfied!
I was immediately drawn into this story. Definitely worthy of the awards it has received. I have found few books as satisfying as this one. It tells a gripping story with language that is real and meangingful. There is no gimmicktry of plot device, no sense that the author is trying to make it happen. To me, it flowed very naturally and seemed to impart reality. I loved that I felt like I could believe this story, which allowed me to learn from it. I am looking forward to reading all of Louise Erdrich's work! She now ranks among my favorite authors along with Sherman Alexi, Jimmy Santiago Baca, Rudolfo Anaya, Isabelle Allende and Barbara Kingsolver.
I was very conflicted while reading this book. The author has great literary talent. But the book was not my kind of book. I really had to plow through it; like plowing a deep snowfall. I kept at it because it was my readers' club selection. First of all there is very little character interaction. The book is mostly a narrative told by the main character. The plot is buried in a mound of Native American history and culture. The plot gets lost. I got absolutely no enjoyment from reading this book.
I read A Plague of Doves before reading this and it was good but this one was better. So many of the characters reminded me of someone I know and it brought the story to life. It's on my recommended reading list.
In a seemingly obvious intersection between Native American Literature and Crime Literature, violence plays a key role in The Round House. Violence, revenge, and kidnapping thrust the reader to the conclusion that violence against Native Americans, and Native American women in particular, is commonplace and often goes unresolved. Worse, many crimes go mostly uninvestigated, much less prosecuted. This gives native peoples the impression that they have no power, rights, or worth and must take matters into their own hands. Violence becomes a vicious circle or hate and a retaliation and a means to an end. The Round House provides a view into this cycle and is the driving point behind the plot and character development. Violence and death are dealt with a familiar and off hand approach in the book. All the characters have grown accustomed to it and understand how it can affect those around them. The characters that would be considered "good" use it as a means to an end with which to punish those responsible for heinous crimes when traditional means fail. Violence against Natives and Native Women in real life is all too common and justice far too rare. The Round House calls into question the validity and the red tape that makes up laws on Native American Reservations and tribal police's ability to prosecute crime and defend their people. It provides a shocking and relatable story about a rape and how it is dealt with by family, community, and the law that appears to be all too common for Native American women of past and present. It calls for a change to tribal laws, one that would increase their tribal law power and their sovereignty on their own land. This would afford them better opportunities to protect their people and help restore the rights taken from them by the government and history.
Captivating, educational, couldn't put it down.
I liked this story. I felt Like I knew the characters. The writing kept me interested.
I loved this book on so many levels...the characters are honest and relatable. The story line has everything..a mystery, a coming of age tale, and social injustice themes. This book makes you think, but more importantly, it makes you FEEL. I was invested in these characters, their lives, their flaws, and their loves. The moral issues are complex and heart wrenching. This book does what a great story should...gives you empathy and perspective for a situation that is beyond anything that most of us experience. I was transported, emotionally challenged, and made aware of social issues that I had not thought existed. Thank you to this author for sharing the lives of these characters with me and opening my eyes to a new perspective and awareness of the native people of our nation.
I love everything about this book.


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subset: commoncrawl


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The lines might be out the door, but Star Wars: The Force Awakens is playing just about everywhere on planet Earth this week. Turns out, the sci-fi epic is also playing on a screen a good bit further away, as well.
British astronaut Tim Peake confirmed that the film should make its way to the International Space Station, and they'll actually get to see the flick on a newly installed projector screen. As for actually getting the movie there, communication to the ISS has come a long way these days, so mission control is more than capable of beaming some movies and TV shows into orbit. Movie night on the station is apparently Saturday, so they could fire up the latest addition a galaxy far, far away as early as next week.
It's awesome to think that even the astronauts in orbit will get a chance to participate in this massive cultural touchstone. Just because they're on the ISS doesn't mean these folks have to be left out of all the cool stuff we're doing down here. Excellent move by Disney to work this one out.
Needless to say — there's not a more fitting blockbuster for these real-life space explorers.
(Via Space)


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Duquesne Graduate Students Earn Phi Kappa Phi Love of Learning Awards
Phi Kappa Phi, the nation's oldest and most selective collegiate honor society for all academic disciplines, has chosen two Duquesne graduate students to receive Love of Learning awards. Linda Ferris and Alexandra Reznik have each been awarded $500.
Ferris is a graduate student in the School of Leadership and Professional Advancement working toward her master's degree in community leadership. Ferris, a program coordinator at Orange County Community College, lives in Montgomery, N.Y. and plans to apply funds from the award toward tuition and books.
Reznik, an Allison Park resident, is a second-year doctoral student in the department of English. She plans to apply funds from the award toward her recent trip to Ghana, which allowed her the opportunity to research culture and literature at Accra, the Spiritan University College in Ijitsu, and Cape Coast.
The Phi Kappa Phi Love of Learning Award program was initiated in 2007 to help fund graduate or professional studies, doctoral dissertations, continuing education, career development and travel related to teaching and studies for active members. The society's award programs provide more than $1 million every two years to qualifying students and members through graduate fellowships, undergraduate study abroad grants, member and chapter awards, and grants for local and national literacy initiatives.
Duquesne University
Founded in 1878, Duquesne is consistently ranked among the nation's top Catholic universities for its award-winning faculty and tradition of academic excellence. Duquesne, a campus of nearly 9,500 graduate and undergraduate students, has been nationally recognized for its academic programs, community service and commitment to sustainability. Follow Duquesne University on Facebook, Twitter and Instagram.


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As someone who has family members with autism-spectrum disorders – indeed, who has one himself – I find the fact that this happened to be utterly disgusting. I realize how disruptive students with these conditions can be, but I have seen similar situations handled with far more tact. It is a commonly held myth that people with autism-spectrum disorders do not exhibit feelings. Rather they exhibit extremes of feelings which means at times they can feel emotions more than the average person. How traumatic, then, might this have been for this student? Note that in the article, the teacher confirmed the story!
We have only just begun to understand these disorders. Most of the progress on understanding these disorders has come in the past 15 years or so and yet there is still a great deal of debate. There was a terrific article in Wired back in March that dispels even more myths about the condition. In recent years there's also been a growing realization that ADD/ADHD is probably an autism spectrum disorder. Both are often seen in conjunction with OCD (obsessive compulsive disorder). The idea that any of these are due to vaccines is unproven. They are very clearly hereditary (one only needs to join my family for Christmas to understand this). I've included some articles on the link between ADD/ADHD and autism from a conference on autism sponsored by the Association for Behavior Analysis attended by one of my students who sent me these:
Richard Simpson (Kansas) on Asperger's syndrome
Richard Foxx (Penn State) on scientifically treating autism
Bobby Newman (Room To Grow) on applied behavior analysis
Finally, some excellent books on both ADD/ADHD and autism are
Driven to Distraction by Hallowell and Ratey
The Curious Incident of the Dog in the Night-Time by Mark Haddon


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Limited antigenic diversity of Plasmodium falciparumapical membrane antigen 1 supports the development of effective multi-allele vaccines
- Ulrich Terheggen1, 2,
- Damien R Drew1,
- Anthony N Hodder3,
- Nadia J Cross1,
- Cleopatra K Mugyenyi4,
- Alyssa E Barry3, 5,
- Robin F Anders6,
- Sheetij Dutta7,
- Faith HA Osier4,
- Salenna R Elliott1,
- Nicolas Senn8, 9,
- Danielle I Stanisic3, 8,
- Kevin Marsh4,
- Peter M Siba8,
- Ivo Mueller3, 5, 8,
- Jack S Richards1, 2, 10 and
- James G Beeson1, 2, 10Email author
© Terheggen et al.; licensee BioMed Central Ltd. 2014
- Received: 16 July 2014
- Accepted: 10 September 2014
- Published: 16 October 2014
Polymorphism in antigens is a common mechanism for immune evasion used by many important pathogens, and presents major challenges in vaccine development. In malaria, many key immune targets and vaccine candidates show substantial polymorphism. However, knowledge on antigenic diversity of key antigens, the impact of polymorphism on potential vaccine escape, and how sequence polymorphism relates to antigenic differences is very limited, yet crucial for vaccine development. Plasmodium falciparum apical membrane antigen 1 (AMA1) is an important target of naturally-acquired antibodies in malaria immunity and a leading vaccine candidate. However, AMA1 has extensive allelic diversity with more than 60 polymorphic amino acid residues and more than 200 haplotypes in a single population. Therefore, AMA1 serves as an excellent model to assess antigenic diversity in malaria vaccine antigens and the feasibility of multi-allele vaccine approaches. While most previous research has focused on sequence diversity and antibody responses in laboratory animals, little has been done on the cross-reactivity of human antibodies.
We aimed to determine the extent of antigenic diversity of AMA1, defined by reactivity with human antibodies, and to aid the identification of specific alleles for potential inclusion in a multi-allele vaccine. We developed an approach using a multiple-antigen-competition enzyme-linked immunosorbent assay (ELISA) to examine cross-reactivity of naturally-acquired antibodies in Papua New Guinea and Kenya, and related this to differences in AMA1 sequence.
We found that adults had greater cross-reactivity of antibodies than children, although the patterns of cross-reactivity to alleles were the same. Patterns of antibody cross-reactivity were very similar between populations (Papua New Guinea and Kenya), and over time. Further, our results show that antigenic diversity of AMA1 alleles is surprisingly restricted, despite extensive sequence polymorphism. Our findings suggest that a combination of three different alleles, if selected appropriately, may be sufficient to cover the majority of antigenic diversity in polymorphic AMA1 antigens. Antigenic properties were not strongly related to existing haplotype groupings based on sequence analysis.
Antigenic diversity of AMA1 is limited and a vaccine including a small number of alleles might be sufficient for coverage against naturally-circulating strains, supporting a multi-allele approach for developing polymorphic antigens as malaria vaccines.
- Malaria
- Plasmodium falciparum
- Vaccines
- Immunity
- Apical membrane antigen 1
- Cross-reactive antibodies
Malaria continues to have a profound impact on the health of children and adults around the world, causing approximately 219 million clinical cases and 660,000 deaths per year [1]. Even though the scale-up of malaria-control interventions has resulted in a notable reduction in morbidity and mortality over the last decade, drug resistance remains a significant concern, and a cost-effective vaccine could play a major role in control and eventual elimination of the disease.
Understanding the mechanisms by which naturally-acquired immunity to malaria protects against death and severe disease is important for informing the rational design and development of effective vaccines. Antibodies constitute a major component of naturally-acquired immunity [2]-[4]. The merozoite form of the parasite, which invades red blood cells, expresses antigens that are prominent antibody targets [5]. Merozoite antigens are attractive vaccine targets because antibodies to these antigens inhibit red blood cell invasion and promote opsonic phagocytosis and antibody-dependent cellular inhibition that limit blood stage replication and prevent disease [6]-[9]. Furthermore, antibodies to merozoite antigens are associated with protection from malaria [10], and several merozoite antigen-based vaccines have shown protective efficacy in animal models [6],[11]. A major challenge in developing merozoite-based vaccines, and other vaccines based on antigens that are targets of natural immunity, is overcoming potential antigenic diversity. Most major immune targets, and many vaccine candidates, show substantial polymorphism in sequence that have evolved to facilitate immune evasion. Vaccine approaches are needed to account for this polymorphism such that they will cover the majority of strains causing infection and disease. Although sequence polymorphism has been described for many antigens, knowledge is very limited on the extent of antigenic diversity (defined by antigen reactivity to human antibodies) and how polymorphisms relate to antigenic diversity for most leading candidate antigens, yet this is crucial for advancing vaccine development.
There are more than 40 different merozoite antigens on the surface or in the apical organelles of merozoites, few of which have been investigated as immune targets [5],[6],[10]. One important target, and leading vaccine candidate, is apical membrane antigen 1 (AMA1), which plays an essential role in erythrocyte invasion [12]. Antibodies to AMA1 are highly prevalent in malaria-exposed individuals and their prevalence increases with age as naturally-acquired immunity develops [13]. Antibodies to AMA1 have been associated with reduced risk of clinical malaria in prospective studies [14]-[16] and in vitro data indicate that AMA1 antibodies can inhibit parasite invasion of erythrocytes [17]-[19]. AMA1 is a promising blood stage vaccine candidate which is presently being tested in clinical trials. A recent phase II trial of a monovalent AMA 1 vaccine in one- to six-year-old children in Mali showed 65% strain specific efficacy [20]. However, AMA1 is a highly polymorphic protein with more than 60 polymorphic sites and more than 200 haplotypes per population [21],[22], one of the most polymorphic of all merozoite antigens. Immunization with one allele of AMA1 may not protect against parasites expressing different AMA 1 alleles, as highlighted by the Mali trial; there was no overall protection against clinical malaria, but there was evidence of protection against malaria caused by vaccine-like alleles [20]. While sequence analysis has been used to classify AMA1 alleles into related groups that might show cross-reactive immunity, the antigenic diversity of AMA1 and cross-reactivity of antibodies are poorly understood, and it is unclear how sequence polymorphisms and sequence-based groupings relate to antigenic diversity and escape from acquired human antibodies. There are only limited data on antigenic diversity in human studies and limited data to understand how sequence diversity is related to antigenic diversity, which is an impediment to vaccine development. Understanding these issues is essential for advancing AMA1-based vaccines. AMA1 also serves as an ideal model to examine antigenic diversity more broadly, the significance of polymorphism in vaccine development and the feasibility of developing multi-allele vaccines based on polymorphic antigens.
We sought to define antigenic diversity of AMA1 and use this knowledge to understand which AMA1 alleles could be included in a multi-allele vaccine to achieve the broadest coverage of AMA1 diversity, and establish principles that could be applied to other polymorphic vaccine antigens. Antibody reactivity to various geographically-diverse AMA1 alleles was examined among children of different ages and adults from two geographically diverse malaria endemic regions (Papua New Guinea and Kenya). We examined the relationship between antigenic diversity and sequence diversity, and sought to establish whether overall antigenic diversity of AMA1 is limited and might ultimately be reduced to a small number of major serotypes. To achieve these objectives, we developed a novel approach that we named multiple antigen competition enzyme-linked immunosorbent assay (ELISA) (MACE) that can also be used to define antigenic diversity of other polymorphic antigens.
Study cohorts and sample collection
Serum samples were collected from a cross-sectional study in Madang Province, Papua New Guinea (PNG) in 2007, and included 118 individuals: 49 adults (median age 28 years) and 69 children (median age 6 years). From these samples we prepared pools of AMA1 antibody positive samples for testing in competition ELISAs; one pool was made from children's samples (n = 31; median age 7 years (range 4 to 10)) and one from adult samples (n = 42; median age 28 years (range 16 to 53)). To prepare the pools, all sera were first tested in standard ELISA for immunoglobulin G (IgG) reactivity against five different recombinant AMA1 alleles (3D7, W2mef, FVO, 7G8, and HB3). After screening, we excluded antibody negative samples and samples with low antibody reactivity (defined as below the 25th centile). All samples included in the pools were antibody positive to all five AMA1 alleles, and antibody reactivity to different alleles was highly correlated (>0.9 for all comparisons), as we have found previously in this population [16]. In preparing pools, an equal volume of all individual samples was used, and substantial numbers of samples were included in pools to account for variation in antibody levels and cross-reactivity among individual samples.
Samples were also obtained from an extended longitudinal study in Kilifi, Kenya (Ngerenya cohort). More than 300 children were included in the original Kenyan cohort [23]; in our study we examined serum from a subset of 42 children who were positive for AMA1 antibody responses and were present for screening and sample collection at two different time points, October 2002 and October 2004. From these samples we prepared a pool of AMA1 antibody positive samples for testing in competition ELISAs using samples collected from the same children at the two different times. To select samples for inclusion in the pools, individual samples were first screened for IgG reactivity to three AMA1 alleles (3D7, W2mef and HB3) in standard ELISA. The levels and proportion of positive samples were lower than for the PNG samples. For preparation of pools we selected the top quartile of responders to AMA1-3D7; however, antibodies to different AMA1 alleles were highly correlated (correlation co-efficient 0.77 to 0.95) and all individuals had antibody reactivity greater than the group median for all AMA1 alleles. The median age (range) of children in the sample pools was 6.5 years (2.1 to 7.6) for October 2002, and 8.3 years (4.2 to 9.8) for October 2004. Sera from unexposed Australian blood donors donated by the Red Cross Blood Bank were used as negative controls in ELISAs and antibody positive samples were defined as those with reactivity greater than the median + three standard deviations (SD) of the Australian controls. Ethical approval was granted by the Kenya National Research Ethics Committee, the Medical Research Advisory Council PNG, the Walter and Eliza Hall Institute, and Alfred Hospital Human Research and Ethics Committees. Written informed consent was obtained from all participants or their guardians.
AMA1 alleles
P. falciparum AMA1 alleles selected for this study
Preparation of recombinant AMA1
Recombinant AMA1 proteins used in this study were expressed, purified and refolded using established protocols [19],[24],[36]. Nucleotide sequences were amplified from genomic DNA using Pfu DNA polymerase and oligonucleotide primers. The amplified products were digested with BamHI and Xho1, and ligated into pProEXHT-B 6xHis, and transformed into Escherich i a coli strain BL21. (Proteins were solubilized in 6 M guanidine-HCL, which completely denatures the recombinant proteins). After purification on nickel resin, AMA1 protein was refolded with reduced and oxidized glutathione redox pairs. Refolded AMA1 was further purified by anion exchange chromatography, followed by reversed-phase, high performance liquid chromatography (RP-HPLC). Refolded AMA1 was identified by a shift in the monomer peak on RP-HPLC and a migration shift on SDS-PAGE when compared to a reduced sample of the refolded AMA1 preparation. Full details of preparation can be found elsewhere [19].
Competition ELISAs
Standard ELISAs to measure IgG to recombinant AMA1 were performed using established methods. Serum samples were tested in single and multiple antigen competition ELISA (MACE) against 11 AMA1 alleles. A novel method, MACE, was developed by modifying the conventional competition ELISA assay to allow cross-reactivity among several alleles of one protein to be examined. In a conventional competition ELISA assay, human antibodies are pre-incubated with a single competing antigen before adding to wells coated with a different allelic variant of the same antigen and proceeding as for a standard ELISA. In MACE, human antibodies are pre-incubated with combinations of up to four competing heterologous recombinant alleles. A total of 75 different combinations were used for competition. The level of inhibition in the presence of one or more competitor alleles indicates the degree of cross-reactivity with the tested allele used to coat the wells [see Additional file 1: Figure S4].
ELISAs were carried out using 96-well polystyrene Maxisorp plates (Nunc, Roskilde, Denmark) coated with one of eleven recombinant AMA-1 proteins at a concentration of 0.5 μg/ml in phosphate-buffered saline (PBS) and were incubated overnight at 4°C. Plates were blocked using 0.1% casein in PBS-tween 0.05%. Plasma was diluted to a concentration of 1:1,000 and one or combinations of several competing recombinant proteins (AMA-1 variants) were added in excess, each at 5 μg/ml. Prior to performing competition ELISAs, we optimized the conditions of our assays to ensure that antibody reactivity with pools was below the saturation point and the serum concentration gave an Optical Density (OD) reading at 405 nm that was in the linear part of a titration curve showing the relationship between antibody concentration and OD (for example, see [37]). Optimization included titration of serum dilutions and antigen coating concentration, and optimization of secondary antibody concentrations and incubation times for the substrate. Additionally, we titrated the concentration of the competitor antigen to determine the concentration required to fully saturate antibody binding and achieve maximal inhibition. A concentration 5 μg/ml of competition antigen was well above the saturation threshold and was used in all competition ELISAs [see Additional file 1: Figure S3). For each of the 11 AMA-1 variants used as coating antigen, the homologous allele and the heterologous recombinant AMA-1 antigen combinations were added as competitor. The homologous competitor served as internal control for competition [see Additional file 1: Figure S4). The plasma/competitor mix was allowed to pre-incubate at room temperature for 30 minutes and then added to the plate (following washing) for 2 hours. Incubations were done using 50 μl volume per well in duplicate and washes between each incubation were carried out using PBS-tween 0.05%. All incubations occurred at room temperature. To determine total IgG to plate-bound antibodies, horseradish peroxide (HRP)-conjugated sheep anti-human IgG (Millipore, North Ryde, Australia) at 1 in 5,000 was used and allowed to incubate for one hour. ABTS (2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)) substrate (Sigma-Aldrich, Castle Hill, Australia) was added to develop color and the reaction was stopped using 1% sodium dodecyl sulfate (SDS) after 20 minutes in the dark. The optical density of each sample was measured at 405 nm (ABTS) using a plate reader (Thermo Fisher Scientific, Scoresby, Victoria, Australia). The mean of duplicate wells was calculated and background (wells coated with antigen and incubated with PBS plus competitor) was subtracted for each sample (using ScanIt MulticanPro software). Results on plates were standardized to adjust for plate-to-plate variance using positive control pools on each plate tested [16], and the mean plus three standard deviations of the OD for Australian negative control sera was used as the cut-off for seropositivity.
Data analysis
Cross-reactivity was defined as degree of inhibition of antibody reactivity by one or more competitor. Standardized OD values were used for calculating the proportion of AMA1 specific reactivity to the coating AMA1 allele that was competed by the heterologous competitor(s) ((heterologous competition - homologous competition)/(no competition - homologous competition) × 100) [see Additional file 1: Figure S4]. ClustalWeb 2.1 was used for amino acid sequence alignments. Statistical analysis was performed using GraphPad Prism 5 (Graphpad Software). Correlations between cross-reactivity and sequence polymorphisms between AMA1 alleles were calculated using Spearman's rank correlation coefficient (ρ). Two-tailed P values were calculated for each rho to determine the significance of correlation. Wilcoxon's rank sum test was used to compare the level of antibody cross-reactivity between groups, except for comparisons of Kenyan children at two time points for which the Wilcoxon's signed rank sum test was used.
To investigate phylogenetic relationships among AMA1 sequences, a network analysis was performed for 873 AMA1 ectodomain sequences (residues 148 to 553) available from GenBank including the 11 reference alleles used in the present study. A second network analysis was performed restricted to sequences available from our study populations: this included 49 sequences from Chonyi, Kilifi District, Kenya and 31 from Mugil, Madang Province, PNG. Network analysis was performed using Phylogenetic Network software version 220.127.116.11 together with the add-ons DNA Alignment and Network Publisher (Fluxus-engineering, Germany). The network analysis was based on the Median Joining algorithm which connects haplotypes on the basis of the numbers of shared alleles and allows for the multiple connections between haplotypes arising through recombination. Gene accession numbers: KF698984 to KF699059 and FN869649 to FN869697.
Antibody cross-reactivity to different AMA1 alleles
Competition ELISAs with human antibodies were used to examine antigenic diversity of AMA1 and investigate whether strain-specificity of human antibody responses to AMA1 could potentially be overcome with the inclusion of a limited number of alleles in a multi-allele vaccine. Competition ELISAs were required because measuring antibodies to different AMA1 alleles by standard ELISA does not sufficiently discriminate the levels of cross-reactivity and allele-specificity of antibodies. AMA1 alleles of diverse geographical origins were selected (Table 1). Phylogenetic tree analysis has shown previously that these alleles broadly represent global AMA1 diversity [24]. In order to efficiently test the many antigen competition comparisons in this study, we prepared a pool of serum samples from PNG children (n = 31; median age 7 years) and a pool of adult samples (n = 42; median age 28 years) selecting those who were antibody positive after initial screening for reactivity to AMA1 in standard ELISA. We confirmed the use of the pools as an appropriate strategy by demonstrating that competition ELISA results using pools revealed very similar patterns and levels of antibody cross-reactivity as those obtained when testing all samples individually [see Additional file 1: Figure S5]. We have previously reported the use of pools to measure other anti-malarial antibodies [38].
Greater cross-reactivity of antibodies among adults compared to children
Patterns of cross-reactivity are similar over time and between different populations
Multiple antigen competition ELISAs
In some assays, we found that a combination of three heterologous alleles could compete almost as successfully as the homologous allele, indicating that most antibodies to the coating AMA1 allele cross-reacted with one or more of the competitors (for example, 3D7, W2mef and HB3 in Figure 5; Additional file 1: Figures S7 and S8). In contrast, for other combinations the addition of the third heterologous allele showed little additional benefit over two competitors, most likely because antibodies reactive with this allele were already binding the other competitor alleles (for example, adding HB3 to 3D7 + FVO, Figure 5B1 and B2). These data were generated using sera from PNG children, but similar results were observed in PNG adults [see Additional file 1: Figure S6]. The complete data set of all double and triple combinations in PNG children can be found in Additional file 1: Figure S7. Multiple competition ELISAs with combinations of two or three alleles were also performed with sera from the Kenyan cohort, with similar results [see Additional file 1: Figure S8]. Once again, similar patterns of cross-reactivity were observed in the PNG and Kenyan cohorts, with slightly higher cross-reactivity in Kenya. The combination of three alleles that showed the broadest cross-reactivity in PNG (3D7, W2mef and HB3) also showed the greatest coverage in Kenya. Not only have we found the same patterns of cross-reactivity in both geographical regions, but also that the same combinations may provide almost complete coverage in terms of cross-reactivity in both cohorts (Figure 5 and Additional file 1: Figure S8). These findings provided the rationale for further analysis to identify combinations of alleles that might cover antigenic diversity of AMA1 that could guide the selection of alleles for inclusion in multi-allele vaccine development.
Identifying combinations with broad coverage as possible multi-allele vaccine candidates
Sequence diversity in AMA1 alleles
Relationship between sequence differences in AMA1 alleles and antibody cross-reactivity
Many targets of naturally-acquired immunity to malaria and leading vaccine candidates are polymorphic, which presents challenges to developing these antigens as vaccines that generate broadly protective immunity against different circulating variants or strains in populations. Understanding the antigenic diversity of polymorphic antigens, the level of allele-specific and cross-reactive antibodies, and the relationship between sequence polymorphisms and antigenic escape are essential for advancing vaccine development; however, this knowledge is currently limited for polymorphic malaria antigens. An example of such an antigen is AMA 1, which is a leading malaria vaccine candidate [20],[39] and also one of the most polymorphic merozoite antigens. Given the extensive diversity of AMA1 in most populations, single allele immunization is likely to be of limited benefit in endemic areas [20],[22]. Here, we investigated antigenic diversity of AMA1 (defined by reactivity to human antibodies) to determine strategies for the development of a potential multi-allele AMA1 vaccine, and more broadly as a model of polymorphic malaria vaccine candidates and the multi-allele vaccine approach. We investigated the antigenic cross-reactivity of a geographically diverse panel of AMA1 alleles with the aim of identifying alleles that might be included in a broadly protective, multi-allele vaccine. Antigenic diversity of AMA1 was found to be surprisingly restricted, despite the large number of haplotypes present in populations. Extensive antibody cross-reactivity against different AMA1 alleles was observed, and results from multiple antigen competition ELISAs indicated that a three-allele vaccine may be sufficient to provide broad coverage against naturally circulating strains, provided the correct alleles are selected. Earlier data from Osier et al. indicated that high levels of antibodies to only three AMA1 alleles measured by ELISA were strongly associated with protection from clinical malaria [41]. These results were suggestive that co-acquisition of different allele specific antibodies may produce cross-protection to a larger number of naturally-circulating strains. Until now, there has been little direct evidence to support this interpretation. Our study on cross-reactivity of human anti-AMA1 antibodies helps resolve this question, defining the extent of cross-reactivity towards different alleles.
Competition ELISA experiments demonstrated that antibodies to AMA1 alleles were extensively cross-reactive. Cross-reactivity of more than 70% was observed for some pairs of alleles, suggesting high levels of antigenic relatedness. While other pairs of alleles showed lower cross-reactivity, all combinations displayed at least some cross-reactivity (minimum was 10%). Antibodies that cross-react against different AMA1 alleles probably target shared epitopes (common epitopes that are shared across different alleles) rather than target strictly conserved epitopes (epitopes that are identical on all alleles). Cross-reactive antibodies that protect against different strains of the same pathogen have been well studied in viral diseases, such as influenza or dengue [42]-[44], and less so in bacteria [45], but there are only limited data on cross-reactive antibodies against individual P. falciparum antigens (for example, [31],[46]). This is the first study to comprehensively examine the extent to which naturally-acquired human AMA1 antibodies can react with multiple different AMA1 alleles and the most comprehensive analysis of the antigenic diversity, defined by human antibody reactivity, to any merozoite antigen. Although antigenic diversity to AMA1 has also been assessed through the use of antibodies generated in rabbits by immunization, these responses may not be entirely representative of human responses and may differ in nature and specificity. While studies of responses generated by immunization in animal models are informative, reactivity of human antibodies is the most relevant response to evaluate antigenic diversity of AMA1. In addition, the antigenic diversity of AMA1 has been largely evaluated with growth inhibition assays in published studies [24],[35],[40], and it is not clear whether this is the primary effector mechanism mediating protection. Therefore, we took an approach to measure human antibodies to all epitopes on AMA1.
Competition ELISA experiments comparing cross-reactivity of AMA1 antibodies among malaria-exposed adults and children showed greater antibody cross-reactivity in adults, reflecting a higher level of protective immunity in adults. This observation is supported by an earlier cross-sectional study of AMA1 antibody responses in PNG [13] that showed that of the few individuals identified as having highly allele-specific responses, most were children younger than 10 years of age; however, they did not directly assess antibody cross-reactivity in children versus adults. If conserved or cross-reactive epitopes are less immunogenic than polymorphic epitopes, antibodies might not be produced by younger children either because their immune systems are functionally immature or they have had insufficient exposure to the antigen. Another explanation is suggested by animal immunization studies, which showed that a higher proportion of cross-reactive antibodies to AMA1 was induced by immunization with multiple alleles compared to immunization with a single allele [47],[48]. In humans, acquisition of cross-reactive antibodies may also require exposure to a range of alleles, which will be reflected in an increase in the relative proportion of cross-reactive antibodies with cumulative exposure. Future studies to understand variation in the patterns and levels of antibody cross-reactivity to different AMA1 alleles among individuals, and how that relates to age and exposure, will be important and may further inform the selection of alleles for inclusion in a possible multi-allele vaccine. Further studies using an expanded repertoire of AMA1 alleles in single and multiple competition ELISAs, and in additional populations, may also help refine allele-selection in vaccine design.
Importantly, we found very similar patterns of cross-reactivity and allele specificity when comparing AMA1 antibody responses in geographically distinct populations. This suggests that different populations are exposed to a similar repertoire of AMA1 alleles, and that the antigenic properties of AMA1 alleles are the major determinants of cross-reactivity between alleles rather than differences between populations. Furthermore, it is likely that exposure to multiple different alleles in naturally-acquired infections will also influence the acquisition of cross-reactive antibodies. Consistent with our observations, sequence analyses have indicated that while sequence diversity of AMA1 is high, most of the diversity found globally can be identified within a single geographical location [35],[49], although there is some geographic clustering of alleles. Analyses suggested that the 11 alleles included in our studies were broadly representative of global diversity. Encouragingly, our findings suggest that the same multi-allele AMA1 vaccine could be effective in different regions and populations.
Multiple competition ELISAs with mixtures of three competitor alleles suggested that antibodies represented in two allele combinations, Combination A (3D7, W2mef and HB3) and Combination E (D10, W2mef and FVO) gave broad, high level cross-reactivity in two geographically distinct human populations (Figure 6), suggesting that a multi-allele-vaccine could probably be reduced to either of these combinations of three major AMA1 serotypes. We propose that the novel MACE approach we have used here could be adopted as an efficient and cost-effective means of testing any polymorphic antigen for its antigenic diversity and the potential of multi-allele approaches in vaccine development. Results from competition ELISA experiments could be used to allocate alleles into potential antigenic or serologic groups that could guide allele selection for AMA1 vaccine development. Alleles with high cross-reactivity against each other by single competition ELISA would be considered to belong to the same serogroup. When two competitor alleles from the same serogroup were combined in double-competition ELISAs, cross-reactivity would not be greatly enhanced compared with results when each allele was used alone as a competitor, reflecting antigenic relatedness between the alleles (see Figure 5). In contrast, combining two competitor alleles from different serogroups would increase cross-reactivity against different alleles. This process guides the selection of alleles to use in combinations.
Several studies have described AMA1 sequence diversity and generated allele-clusters based on sequence similarities using the Structure algorithm [22],[35],[49], with some evidence that these groupings are immunologically relevant. The current study represents the first time AMA1 groupings have been compared to antigenic diversity defined by human antibodies, and we were able to examine how antigenic diversity related to sequence diversity. While there was some relationship between the number of sequence differences between two alleles and the level of antibody cross-reactivity, at times we observed high antibody cross-reactivity between two AMA1 alleles despite a high number of amino acid sequence differences. Our data indicate that broadly assessing sequence differences by the extent of sequence identity between alleles was not a strong or reliable predictor of antibody cross-reactivity between AMA1 alleles, and antigenic diversity is more restricted than sequence diversity. Similarly, grouping sequences using the Bayesian clustering approach used in Structure[22],[35],[49] was also not a strong or reliable predictor of antigenic differences. The lack of a strong relationship between overall sequence differences or haplotype clusters and antigenic differences suggests that only a subset of all polymorphisms are important for defining antigenic differences, that groups of polymorphisms are required for significant antigenic differences, or that some polymorphisms play only a minor role in influencing antigenic differences between alleles. For some other polymorphic malaria antigens, results have suggested that antigenic diversity can be relatively restricted despite significant sequence diversity (for example, EBA175, var2csa [31],[50],[51]). A previous study identified a polymorphic cluster (C1-L) in AMA1 important for escape from vaccine-induced rabbit antibodies; we found that differences in this region, on their own, were not a good predictor of the antigenic differences we measured here (data not shown). Identifying the most important residues that determine antigenic differences and facilitate immune escape is an important focus for future research; this knowledge would enable the development of sequence-based algorithms that better predict antigenic properties of alleles and would be valuable for application in AMA1 vaccine trials. Recent data from growth inhibition assays using rabbit antisera raised against different AMA1 alleles [24] support these conclusions, demonstrating that the extent of sequence identity between alleles was weakly and inconsistently predictive of antibody cross-inhibitory activity in growth inhibition assays. Furthermore, a mixture of antibodies to four AMA1 alleles was sufficient to inhibit growth of a diverse array of P. falciparum isolates [24]. Detailed evaluation of antigenic diversity is crucial for defining which alleles should be included in a multi-allele AMA1 vaccine.
AMA1 antibodies examined here were acquired from natural exposure, and future studies are needed to further evaluate the specificity and cross-reactivity of responses generated by human immunization to support the multi-allele vaccine approach. Immunization of rabbits with a mixture of AMA1 alleles induced a relatively greater proportion of broadly cross-reactive antibodies than single allele immunization [47]; multi-allele immunization appears to shift the antibody responses to epitopes that are conserved or have limited diversity and, therefore, antibodies have greater cross-reactivity than those generated by single-allele immunization [48]. This provides supporting evidence for the multi-allele vaccine approach. However, whether this finding holds true in humans using less potent adjuvants needs to be determined. Supporting these findings, a prior study demonstrated good immunogenicity of a multi-allele AMA1 vaccine in macaques [52]. This vaccine was comprised of AMA1 proteins based on three synthetic sequences (DiCo) that aimed to cover sequence diversity; vaccine-induced antibodies had similar reactivity by ELISA to four different alleles tested (FVO, HB3, 3D7 and CAMP alleles) and gave significant growth inhibition against the three isolates tested (FCR3, HB3 and NF54). Another rabbit immunization study [40] used mixtures of recombinant AMA1 proteins representing each of the cluster groups defined by Duan et al. [35] and showed that a five-allele mixture was sufficient to generate antibodies with broad growth inhibitory activity against different isolates. Our findings are in general agreement with these results and, given the differences between humans and experimental animal models in nature and specificity of antibodies, our study provides valuable additional information to guide selection of allele-combinations for human trials and predict vaccine coverage in malaria-endemic settings rather than relying only on small animal immunization studies; findings from different approaches need to be used together to inform vaccine design.
A bi-allele vaccine consisting of recombinant 3D7 and FVO AMA1 alleles was shown to induce antibodies to both alleles in a clinical trial [53],[54]. Vaccine-induced antibodies showed cross-reactivity against a non-vaccine AMA1 allele, although the magnitude of response was lower than that against vaccine alleles [53]. Unfortunately, this vaccine showed no efficacy in a phase II trial in Malian children [55]; subsequent investigation found no evidence of strain-specific efficacy (although the sample size was small), and it was suggested that the lack of efficacy was probably because the vaccine formulation was insufficiently immunogenic [56]. Interestingly, the combination of 3D7 and FVO alleles (as used in this vaccine) showed only limited cross-reactivity against different alleles in our study, highlighting the need to carefully select alleles for inclusion in a multi-allele vaccine, with consideration to be given to antigenic relatedness as well as prevalence of circulating alleles in the target population.
Although antibodies measured by ELISA, as performed in our studies, do not directly assess antibody function, they do appear to be a good correlate of functional activity. Antibody levels by ELISA correlate with growth-inhibitory activity in animal and human studies [19],[36],[40], and antibodies to functional invasion-inhibitory epitopes of AMA1 correlated with total antibody reactivity to AMA1 by ELISA [36]. Equally, levels of antibody reactivity to circumsporozoite proteins measured by ELISA appear to be a good correlate of protection with the RTS,S vaccine [57],[58]. Correlations of protection for AMA1 vaccines are not yet available. Further studies that are able to demonstrate comparable levels of cross-reactivity between functional human antibodies to AMA1 alleles (for example, using growth inhibition assays) would provide additional support for the development of a multi-allele AMA1 vaccine, and further research to define the functional activity of AMA1 antibodies is needed. The extent to which human immunization with a multi-allele AMA1 vaccine could protect against naturally circulating P. falciparum strains needs to be formally assessed in clinical trials; however, our approach represents a useful tool to predict this coverage.
In conclusion, AMA1 has been a strong vaccine candidate for many years, but its extensive sequence polymorphism and the limited efficacy of mono- or bi-valent formulations in clinical trials has presented challenges for developing it as an effective vaccine. It has not been clear whether protection would need to be provided by a large repertoire of allele-specific antibodies or antibodies that are able to cross-react with multiple alleles. Here, we have shown extensive cross-reactivity of naturally-acquired antibodies between alleles of AMA1. Whereas sequence polymorphism in AMA1 is high, antigenic diversity is surprisingly restricted, suggesting the feasibility of a multi-allele AMA1 vaccine. Selected combinations of three AMA1 alleles showed high-level, broad cross-reactivity against a range of AMA1 alleles. While further studies with additional alleles are needed to consolidate our findings and further define the alleles for vaccine inclusion, and subsequent testing of allele combinations in clinical trials is needed, our results support the development of AMA1 as a multi-allele vaccine, possibly in combination with other antigens to maximize protective efficacy against malaria. These findings are also broadly relevant to numerous malaria vaccine candidates that are polymorphic, supporting the concept of multi-allele vaccines as a feasible approach and indicating that antigenic diversity may be much lower than predicted by sequence analyses. Our novel MACE method to define antigenic differences and serogroups could be valuable for examining other polymorphic antigens, to characterize antigenic diversity and assess the potential of antigens as future vaccine candidates.
Authors' contributions
UT, JGB designed the study; UT, NJC, DRD, CKM, performed experiments; UT, JSR, AEB, JGB performed analysis and interpretation of data; DRD, ANH, AEB, FHAO, KM, CKM, RFA,SD, IM, PMS, DIS, NJC, SRE, and JGB contributed reagents, clinical samples and data, and AMA1 sequences; UT, JGB, and JSR wrote the manuscript with contributions from DRD, ANH, AEB, RFA, SD, KM, DIS, NS, and SRE. All authors read and approved the final manuscript.
Additional file
We thank all study participants and staff at the Papua New Guinea Institute of Medical Research, Madang, and Kenya Medical Research Institute, Kilifi, Linda Reiling for help with ELISAs, and Stephen Rogerson, Colleen Woods, Ashley Birkett, Carter Diggs, Mick Foley, and Alan Cowman for helpful comments, and Tom Williams for help with Kenyan samples and parasitemia data. Funding was provided by PATH Malaria Vaccine Initiative, US Agency for International Development, National Health and Medical Research Council of Australia (project grant, program grant and senior research fellowship to J. Beeson; project grant to A. Barry, training award to J. Richards; Infrastructure for Research Institutes Support Scheme Grant), Australian Research Council (Future Fellowship to J. Beeson), Victorian State Government Operational Infrastructure Support grant, and Swiss National Science Foundation (U. Terheggen). This paper is published with the permission of the Director of KEMRI. N. Senn is supported by an academic scholarship `bridge-relève' provided by the Leenaards foundation, Switzerland.
Authors' Affiliations
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SY58294Z - Single Stage Buck PFC Regulator For LED Lighting
- · Integrated 500V MOSFET
- · Valley turn-on of the MOSFET to achieve low switching losses
- · Power factor >0.90 with single-stage conversion.
- · Low start up current: 15μA typical
- · Quick start up <500ms
- · Reliable short LED and Open LED protection
- · Power factor >0.90 with single-stage conversion.
- Compact package: SO8
Typical Application
The SY58294Z is a single stage Buck PFC controller targeting at LED lighting applications. It integrates a 500V MOSFET to decrease physical volume. It adopts the proprietary control architecture to achieve an accurate regulation of LED current, unity power factor, and quasi-resonant valley turn-on high efficiency operation. It adopts special design to achieve quick start up and reliable protection for safety requirement.
It integrates open/short LED protection and eliminates the need for opto-coupler, thus minimizing the component count and board size.
- LED lighting


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Steve Francis Provides Yao Ming, Tracy McGrady, & Houston Rockets With Third Scorer
The Franchise is returning to the place where it all began-Houston … Today, Steve Francis inked a two-year deal with the Rockets; he should take some of the scoring load off of Yao & T-Mac … Click here for news and analysis on this breaking story …
ESPN The Magazine reports Steve Francis has signed with Houston:
Steve Francis is going home.
After three years of struggles in Orlando and New York, the former All-Star guard will return to the place of his greatest glory, the Houston Rockets.
Francis was in Houston on Thursday to sign a two-year contract worth roughly $6 million with the Rockets. The club will announce the signing at a news conference on Friday.
"We're extremely pleased that he's going back to Houston," his agent, Jeff Fried, told The Associated Press. "Houston has always been home for Steve since he entered the NBA."
The 30-year-old Francis was recently bought out of the remaining two years, $34 million of his previous contract by the Portland Trail Blazers. He received nearly $31 million in the deal. With money being no factor, Francis chose the Rockets over Miami, Dallas and the Clippers.
My Quick Take: This deal could really benefit Houston and Francis. The Rockets are getting an athletic scorer at a discounted salary, while Francis gets another chance in his second-home.and need a wing-man, a so-called partner in crime. was supposed to fill this role last year, but it didn't work out. Francis, if he accepts his third-banana status, could provide some much-needed scoring for the undermanned Rockets.
Finally, Francis has had a tough go of things. Just a few years ago he was one of the game's best, traded in a package for Tracy McGrady. Now he's receiving left-over crumbs from a buyout. However, the exciting guard is home, back with the team he never wanted to leave. I expect him to thrive and, if all goes well, sign a long-term extension with the Rockets. Maybe then, Francis' famous skip will return to his game.
Click here to read and subscribe to Oly Sandor's NBA Blog.


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Hi strap lovers, Here is a strap that I really love. We just completed it for our good customer David. He had originally ordered a strap we had designed together for his Eterna Kontiki diver, but the acquired a PAM111 and decided to switch up to the Sunrise Ostrich. This one has been hand saddle-stitched using Slate Grey 4 ply waxed linen thread. His original strap strap was designed to include black Kangaroo lining, but that would not work with this strap, because the edges are not painted, but dyed and waxed, so I chose to upgrade his strap using Dangerous9straps' exclusive smoke gray Eel skin lining. I think it's a killer combo with the Sunrise Ostrich and Slate Grey. You'll notice that I show you more than just one photo of my straps but typically provide my customers with between 12 and 20 photos of their completed strap. I do this for a three reasons: 1) I'm proud of my work and and excited about it and want to show it off. 2) I want my customer to be as excited as I am about getting their new strap. 3) I show the backs of the strap because there is something to see there. To me the back of the strap is nearly as important as the front and I really focus on using exceptional and often exclusive materials to make a strap that is a true luxury to wear.
A note about Materials: Dangerous9straps are made using only the very finest materials throughout. Our philosophy is that nothing should go into your strap that I would not be proud to have on the outside. Many straps maker and especially the larger strap manufacturers use foam, cork composites and other lower quality preformed man made materials inside their straps. These materials will breakdown over time and destroy your strap from the inside. Dangerous9straps' mid-layer and padding are made using top grade vegetable tanned leather - the same leather we use to build our leather straps. These leathers will not breakdown over time and as a result, our straps are made to last a lifetime and more.
Here is the completed strap slide show:


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I have been trying to make it to Waffles for about a year or more. I was supposed to meet a friend there for breakfast on a couple of occasions and the dates were cancelled at the last minute. I even purchased a deal for the restaurant and was unable to use it due to a busy schedule. It didn't go to waste, however, as I know a few foodies that can rearrange their schedules for fancy flavors on a whim :). My sister had been to this spot a couple of times and raved about the short ribs and cheddar waffles. I loooove short ribs! So when my good friend, Veronica, came to town and another deal came through, I pounced on it. And I'm so glad I did!
The deal I purchased was good for two entrees and two hot chocolate flights. Oh how I love flights :)! There were sandwiches and other entrees on the menu, but how do you go to a waffle place and not order the waffles? I couldn't fathom it! In addition to the hot chocolate flight, I had the waffle flight (told ya I love flights) and Veronica ordered the short ribs and cheddar waffles. I love to dine with friends and order different things so I can sample, sample, sample! Well, everything we ordered was fantastic! The short ribs were tender little bites of saucy perfection and the cheddar waffles were the perfect accompaniment.
I could not make up my mind as to which of the waffle flavors I wanted to try so the waffle flight was perfect. The flight includes 4 mini versions of the most popular waffles. I was most excited about the green tea waffle with lemon ginger cream. This was a hard choice for me because red velvet anything is my absolute favorite dessert flavor and there are red velvet waffles on the menu. The flight included both of these as well as the liege and mexican chocolate waffles. All of these were great, but I'd have to say the liege and red velvet waffles were my favorites.
I can't wait to go back and try other tempting menu items like the chicken and bacon (bacon waffle w/fried chicken leg and spicy maple syrup), chilaquiles (yummm), and my very own order of short ribs and cheddar waffles (you didn't think I ate all of Veronica's did you?). If you're looking for a great breakfast spot and you love variety like I do (hey, that's why I'm the fickle foodie), give Waffles a try!
Mother of 6, lover of God, life, family, food, friends and flying!