ImmYOUnology

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The most promising treatments for ebola are based on basic immunology-part 2

One of the more peculiar, historic and almost cinematic treatments being discussed in the midst of the ebola crises is the use of blood transfusions. In movies, the blood of a survivor or someone special is often supposed to have some sort of mystical effect on the (usually villainous) recipient. It turns out, blood transfusions from people who have survived ebola are nearly as mystical.

It seemed obvious to me at first that the active components in blood transfusions from ebola survivors must be anti-ebola antibodies. Such antibodies would neutralize the virus and help the immune system clear it out. And in fact, a 1999 study reported that seven patients who survived the 1995 outbreak in the Democratic Republic of the Congo after receiving transfusions from survivors had anti-ebola antibodies circulating in their blood. One kind of antibody, called IgM, was absent in another patient who received a transfusion and died. This very small study seemed to indicate that transfusions could work against ebola and that antibodies are key to making them work. (You may be wondering why, if the transfusions worked, more patients weren’t treated this way during the 1995 outbreak. One important reason is that that blood cannot be transfused unless the donor and recipient blood types are compatible.  So the treatment is limited by the number of willing survivors and their blood types.)

This idea was challenged by a 2007 study done in a nonhuman primate species, rhesus macaques. For this study, researchers drew blood from a rare few monkeys who had survived an ebola infection four or five years earlier and a second “boost” infection 30 days earlier. They transferred the blood into other, recently-infected animals, and none of them survived…even those that made a lot of antibody. There are, of course, caveats. Monkeys are not humans, after all. It is possible that they fight the virus differently. And in the discussion of the paper, the researchers admit the experiments that had successfully transferred antibody-mediated immunity in guinea pigs had not worked in rhesus macaques.

There are also caveats to the human study though. The main one being that it can’t account for the better treatment transfusion patients received compared to other patients. The seven may have survived simply because they received better care in the clinics. The boost of cells, fluids, proteins and electrolytes that come along with blood transfusions may also have helped.

Scanning electron micrograph of red and white blood cells (National Cancer Institute)

Scanning electron micrograph of red and white blood cells (National Cancer Institute)

In spite of it all, the World Health Organization is behind blood transfusions and transfer of plasma from ebola survivors.  Plasma is the liquid part of blood that contains proteins like antibodies, along with other things like electrolytes and hormones. The first of the eight original transfusion patients, a 27 year-old nurse, was originally supposed to receive plasma and not whole blood. This was because, in 1978, a researcher who received plasma survived an ebola infection brought on by a finger prick in the lab. The nurses’ doctors settled for blood because they didn’t have the right tools to separate the plasma (a process called plasmapheresis).

The seven transfusion patients who followed the nurse ranged from a 54 year-old woman to a 12 year-old girl who caught the virus by kissing her newborn niece just days before the infant died. Antibodies seem to be the most likely explanation for the high rate of survival, but it is still not clear whether they were. Well-controlled human trials to determine whether blood transfusions work for ebola will probably never be possible. But, more and more people may be receiving them, so there may soon be more information about whether and how they work.

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The most promising ebola treatments are based on basic immunology

Though for many of us, the ebola crises is oceans away, the epidemic still weighs heavily on the hearts and minds of people all over the world. For some researchers, public health officials and drug developers, it is the driving force of all daily activity. Right now, there are two vaccines and eight treatments being developed or tested for their effectiveness against controlling infection or stopping the  virus’ spread. The most encouraging results have come from treatments that rely on a very basic aspect of immunology: antibodies neutralize viruses.

Antibodies are proteins made by immune cells called B cells. Each one of your millions of B cells is capable of producing antibodies specific for one thing, and when a B cell comes into contact with that one thing, it secretes lots of antibodies. The antibodies then tag invading pathogens, like viruses, to make other immune cells aware of the invader’s presence. If enough antibodies stick to a virus, they can cover it up, or neutralize it, and prevent it from infecting cells.

Ebola infection does trigger an antibody response, but for reasons that are still being studied, those antibodies are not usually enough to stop the virus before it spreads throughout the body. The concept behind ebola treatments like Zmapp, blood transfusions, vaccines and even supportive care, is to help the immune system outpace the growing virus.

 

Zmapp

Over the summer, this product was on headlines everywhere. Zmapp sparked a controversy over who should get the most cutting-edge treatments when it was given to two missionary doctors who flown to Atlanta for care.  Zmapp is not really a drug; it’s a combination of three kinds of antibodies that bind to the surface of ebola virus particles. Because each type was originally produced by one individual B cell they are called monoclonal antibodies. Monoclonal antibodies are used for treating cancer, autoimmune diseases and other infections.

Identifying the right monoclonal antibodies can be a painstaking and years-long process. Researchers collect B cells from a person or animal in the midst of an active immune response, in this case, against ebola. Then they seed individual antibody-making B cells into tiny wells on a cell culture dish. Later they test the culture media from each well for the presence of antibodies and select the cells making the best antibody to be “immortalized.” Cells are immortalized by altering their genes or fusing them with cancer cells that are already immortal. Usually the cells are frozen and stored for later use. They can be thawed anytime and grown to large quantities to make antibody.

Forget cigarettes...tobacco plants have lots of potential for "pharming" biological drugs like the monoclonal antibodies in Zmapp (From Wikemedia Commons)

Forget cigarettes…tobacco plants have lots of potential for “pharming” biological drugs like the monoclonal antibodies in Zmapp (From Wikemedia Commons)

It seems simple, but getting the process right can take years. The monoclonal antibodies in Zmapp were originally derived in mice back in 2000.

From there, the antibodies have to be purified. It can take liters and liters of cell media to purify enough antibody to treat one person one time. As a therapeutic, monoclonal antibodies are typically dosed over multiple treatments. In a recently published study showing the effectiveness of Zmapp against ebola infected monkeys, the animals were treated three to five times a day.

There are alternative ways to do this, however. Because antibodies are proteins they are coded by specific genes. So instead of fusing selected B cells with cancer cells, researchers could copy the gene coding for the cell’s antibody and put it into something else, like bacteria or, in the case of Zmapp, tobacco plants. Many biological products, like insulin have been produced in bacteria since the 1980s. Plant production of human proteins is a bit more recent.  The first human protein produced in plants in 2012 for a medical purpose was an enzyme injected into patients who can’t make it themselves. Some insulin is also now produced in plants.

Unlike cell-based or bacteria-based approaches, plants don’t have to be genetically manipulated and then grown up and harvested. Instead, adult plants are infected with viruses engineered to express the antibody-coding genes. The viruses introduce the genes, and the plants make the antibody. For some proteins, this results in much higher yields than cell-based methods. The monoclonal antibodies in Zmapp are being made by three different companies using a variety of these methods.

But there is a catch. When any kind of cell (plant, animal, bacteria) produces a protein, it adds little sugar labels to keep track of it during each stage of production. This process is called glycosylation. These glyo-labels vary by species and they can affect the way a protein functions. Because of this, the plants being used to grow Zmapp are not your run-of-mill tobacco. They are genetically modified so that they can give the anti-ebola antibodies more human-looking labels. That adds another layer of complexity to be addressed as these companies start to make large quantities of Zmapp.

It’s fascinating how this technology was developed step by step—often in obscurity—over the course of many decades.  Hopefully, it will be scaled up successfully in the coming months to provide more much-needed doses.

Next time…Blood transfusions.


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Allergies are no fun…but the biology behind them is!

Spring is nearly upon us and along with trees and flowers, seasonal allergies will bloom once again.  Even though allergies can be annoying, debilitating and even life-threatening, the science behind them is fascinating.  Science published a timely paper at the end of February describing some of the ways different kinds of allergens work.  Allergens are small parts—individual proteins or molecules—of things that cause allergic responses.

The group who published the study worked with cells called mast cells, one of the common types of the immune cells that respond to allergens and make you itchy, sneezy and swollen.  Before they can activate mast cells, allergens have to be recognized by a particular type of antibody, or immunoglobulin, called immunoglobulin E, or IgE.  On one end, IgE binds an allergen, and on the other it interacts with a protein receptor on mast cell surface.

By connecting the mast cell to the allergen, IgE gives the mast cell permission to do its thing, and its thing is called degranulation.  Mast cells are brimming with packets, or granules, of histamine and heparin and other proteins that damage microbes as well as tissue.  When the cells degranulate, they open up and release their contents into whatever tissue they happen to be in—the skin, the lungs or the gut for example.  Many of the contents released make blood vessels leaky and attract lots of immune cells, causing inflammation.  Antihistamines prevent the released histamine from binding its receptors on blood vessel cells.  Another treatment option currently under investigation is a drug that blocks the interaction between IgE and the receptor on mast cells to prevent this process from even getting started.  

The recent Science paper took a close look at the mast cell response to IgE-bound allergen and showed just how fine-tuned it can be. The researchers activated mast cells with allergens that bound tightly or weakly to IgE and found that the strength of the interaction, also called affinity, changed the way that mast cells responded.

http://commons.wikimedia.org/wiki/File%3ASMCpolyhydroxysmall.jpg

Skin mast cells stained with Toluidine blue

The researchers could study mouse mast cells in culture dishes, because mast cells grow up from stem cells inside bone marrow.  So they grew up mast cells from mouse bone marrow and then gave them the strongly binding allergen (high affinity) or the weakly binding one (low affinity). They could get the mast cells to respond and degranulate with both, but it took 100 times more of the weak binding allergen to get the same response caused by the strong one.

To understand how allergic reactions work in living creatures, researchers often sensitize mouse ears by exposing them to an allergen and later re-introduce the allergen through the bloodstream. Then they can measure how inflamed the ears get and how many and what kinds of immune cells travel to the ear after injecting the allergen.  In this study, the strong binding allergen caused more intense and more sudden ear inflammation and immune cell infiltration than the weaker binding allergen.

So how does this fascinating mechanism actually relate to human allergies, which for some people is a life-threatening condition.  Although some allergies go away with age, there is currently no permanent cure for those that don’t.  Treatment of serious allergies is centered around desensitization immunotherapy, which is just repeated exposure to small doses of allergen over time.  The treatment may last anywhere from months to a lifetime and there are no biomarkers, or biological tests, that tell doctors when the treatment is working.  Instead, they simply test allergens on patients, which could mean pricking the skin or making them eat peanuts one at a time until they do or don’t get sick.  

A clinical study that came out in January helped me understand how knowledge of allergen binding strength could be helpful in treatment.  In this study, children with milk allergies were undergoing oral immunotherapy, which in this case simply meant they had to drink small amounts of milk that were increased over time.  The researchers collected serum samples from the kids in the study and measured levels of IgE as well as the affinity of IgE for proteins found in cow’s milk to see if either would change as kids became more tolerant to milk.

In some cases, the immunotherapy had to be discontinued because the reactions to milk were too severe.  The researchers found that the IgE from the kids whose treatment was discontinued bound more tightly to milk proteins compared to kids who responded well to the treatment.  So the strength of the interaction between IgE and allergens does matter, at least in the case of cow’s milk allergies. This study didn’t look at mast cells, but it does indicate that the molecular details of how IgE connects allergens to mast cells are worth studying.  Those details can provide clues about what is going on inside a person with allergies and how well they may respond to immunotherapy.

Sources:

Mastcellaware.com (A whole website about Mast Cells)

Suzuki R., Leach S., Liu W., Ralston E., Scheffel J., Zhang W., Lowell C.A. & Rivera J. (2014). Molecular Editing of Cellular Responses by the High-Affinity Receptor for IgE, Science, 343 (6174) 1021-1025. DOI:

Savilahti E.M., Kuitunen M., Valori M., Rantanen V., Bardina L., Gimenez G., Mäkelä M.J., Hautaniemi S., Savilahti E. & Sampson H.A. & (2014). Changes in IgE and IgG4 epitope binding profiles associated with the outcome of oral immunotherapy in cow’s milk allergy, Pediatric Allergy and Immunology, n/a-n/a. DOI:

Moran T.P., Vickery B.P. & Burks A.W. (2013). Oral and sublingual immunotherapy for food allergy: current progress and future directions, Current Opinion in Immunology, 25 (6) 781-787. DOI:


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Norovirus! (AKA the 24 hour stomach bug) Can it be avoided?

The other day I found myself in the break room near my lab eyeing a container of chocolate-covered nuts left over from the Christmas holiday.  Someone left them out as a treat for foraging graduate students and post-docs.  I stood for a moment holding a single piece in my fingers and as I was about to put it into my mouth, I remembered—Norovirus!

I had no reason to think the nuts could be a reservoir of norovirus, but I did have good reason to avoid shared uncooked food with an unknown history.   A good chunk of my family had just had their holiday ruined by the virus, sometimes known as the 24-hour bug or stomach flu.  It causes gastroenteritis, or inflammation of the gut, complete with diarrhea, vomiting and overall exhaustion.  It can only be transmitted via stool or vomit, and though there was certainly none of that visible in the bin of delicious looking nuts, I began to think of all the hands that may have been inside. If it came from a family holiday party, some of those hands may have belonged to kids who haven’t yet learned to wash them for a full 30 seconds after using the bathroom. I threw the candy away, closed the container and left the break room.

I may have avoided norovirus that day by a judicious food choice, but not everyone has that moment of doubt before sharing a drink, holding a child’s hand or ordering a deli sandwich.  It is sometimes just unavoidable, especially because it’s contagious for up to two weeks after the first horrible 24 hours. The center for disease control estimates that 19-21 million people are infected with norovirus each year and it’s actually responsible for somewhere between 600 and 800 deaths per year. Those most vulnerable are either over 65 or under 5 years old.

These figures are driving researchers to search for a vaccine, even if just for those most vulnerable or during outbreaks.  But norovirus, or I should say noroviruses are particularly complicated. They are split into 5 groups (I-V) based on how similar their DNA sequences are. Those groups, called genogroups, are split into anywhere between 8 and 30 genotypes and those can be further divided into variants.  The classification is complicated enough to require the use of a software program that compares genome sequences.

Only three of the genotypes can infect humans and the strain GII.4 has been the most common cause of outbreaks since the early 2000s.  For decades before that, a different strain dominated, and the power structure may shift again.  The abundance of genotypes and variants and their changing frequencies in communities make vaccine design a daunting task.  On top of that, researchers are still discovering new genotypes and variants.  In 2012 a strain called GII.4-Sydney was identified in Australia and made its way to the UK and the US within a year.

Norovirus 4

Up close scanning electron microscopic image of norovirus particles

There is evidence that infection with norovirus can generate immunity in some people, meaning that once they get infected, they are protected from re-infection for some weeks or months. However, no one knows how all of the viral subgroups and variants might affect immunity and vaccine design. In a study published in September, researchers from the University of Florida infected mice with one of two closely related norovirus strains and found major differences in the immune responses.

One of the two strains was much better at activating a class of immune cells called antigen presenting cells. These include dendritic cells and macrophages, and they are experts at displaying pieces of virus and training B and T cells to respond to the infection and turn into memory cells. As a result of the enhanced response, infected mice were protected from a reinfection six weeks later.

{Researchers determine “protection” by measuring how much virus shows up in an animal’s organs after infection. In this case, they measured norovirus in the small and large intestines and in the lymph nodes attached to the intestines.}

Oddly enough, the researchers narrowed down the cause of these changes down to a group of structural proteins whose sequences only varied by about 10% between the two strains.

A key finding in this study was that the protective norovirus strain protected mice from re-infection with both strains.  This is important since any vaccine against norovirus would have to protect against several strains and genotypes. It also points out specific characteristics of the immune response that make all the difference between becoming immune or getting re-infected, for example, robust antigen presentation and B and T cell memory.  A vaccine that could foster those characteristics could potentially protect people from several norovirus strains.  It may take a while to get there. In the meantime I will keep my hands clean and out of community candy dishes.

prevent-norovirus

*A reader noted that the poster above says norovirus is contagious for 2-3 days, whereas I wrote above that it can be contagious for 2 weeks.  To clarify, the virus is most contagious for 2-3 days, but it can continue to be shed in stool for 2 weeks. See http://www.cdc.gov/norovirus/preventing-infection.html for more.

Sources:

The CDC

Zhu S., Regev D., Watanabe M., Hickman D., Moussatche N., Jesus D.M., Kahan S.M., Napthine S., Brierley I. & Hunter R.N. & (2013). Identification of Immune and Viral Correlates of Norovirus Protective Immunity through Comparative Study of Intra-Cluster Norovirus Strains, PLoS Pathogens, 9 (9) e1003592. DOI:

Hoa Tran T.N., Trainor E., Nakagomi T., Cunliffe N.A. & Nakagomi O. (2013). Molecular epidemiology of noroviruses associated with acute sporadic gastroenteritis in children: Global distribution of genogroups, genotypes and GII.4 variants, Journal of Clinical Virology, 56 (3) 269-277. DOI:


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A closer look at the immune response to DTaP may explain why it wears off

In my last post, I wrote about how the vaccine against whooping cough or pertussis (the “p” in DTaP) may be wearing off.  Scientists are hard at work characterizing the basics of the immune response to the current acelluar vaccine (DTaP) and the formerly prevalent whole bacteria vaccine (DTP).

What exactly does it mean for a vaccine to “wear off”?  Effectiveness is generally measured by how many vaccinated people get sick.  To follow the immune response to a vaccine, scientists measure immunoglobulin (Ig) levels in the blood.  Ig is made by B cells when these cells detect components either made by bacteria or viruses or engineered into vaccines. Among other things, Ig tags bacteria and viruses as a signal for other cells to attack. As the initial immune response downgrades, B cells that make the strongest-binding Ig are stored as memory B cells or as a different form of B cell called a plasma cell.

Memory B cells wait quietly until they see the same microbe and quickly divide and make large amounts of Ig when they do. Plasma cells wait inside bone marrow and constantly release Ig into the blood as an early defense against any re-exposure to a microbe. Measuring Ig over a long period of time is essentially measuring the health and activity of the plasma cells in the bone marrow.  Ig from both types of B cells help neutralize a re-invading pathogen.

Vaccine protection could wane if the vaccine didn’t produce enough memory B cells or plasma cells, or if cells formed but then quickly died off.  So Ig levels, memory B cells and plasma cells have been common benchmarks to study after vaccination.

Kids enrolled in a Dutch study published in September experienced major drops in pertussis-specific Ig two years after their last booster shots.  But this was true for both the whole bacteria and acellular vaccines.

These results are difficult to interpret because the researchers measured Ig responses to the very three proteins engineered into the acellular vaccine.  The whole bacteria vaccine has a lot more than three proteins that B cells can respond to. So even if Ig levels to the three proteins in the study may be lower, the whole cell vaccine could be inducing an overall higher amount of Ig that is just spread over a larger number of proteins and that information could be missed.

By counting memory B cells from in blood samples the scientist also found that kids given either type of vaccine produced some memory B cells that expanded during the first month after booster but dropped back down by the two year time point. Measuring plasma cells in bone marrow is a bit more challenging in human volunteers, but a study published in 2010 tried to compare these cells in mice after giving them DTaP or DTP.

This group actually found more plasma cells in the bone marrow of the DTaP -vaccinated animals. (Again, the issue of only testing the three antigens found in the DTaP may have skewed these results.) They also found poor memory B cell survival and responsiveness to both forms of the vaccine.

The B cells don’t seem to be acting differently in response to the two vaccines. In fact, the current data suggest that B cells do better after the DTaP, so poor B cell responses are unlikely the main culprit behind the vaccine’s waning protection.

Memory T cells are another force to be reckoned with for infectious bacteria like B. pertussis. The same Dutch study that found better long-term pertussis-specific Ig after the acellular vaccine also saw better T cell responses a year after boost with the whole bacteria vaccine.

An in-depth look at the pertussis-specific memory T cells suggested the whole bacteria vaccine may be better at making memory T cells. Instead of making Ig like memory B cells, memory T cells respond to re-exposure to bacteria or viruses by making immune-stimulating proteins called cytokines.  A group of researchers cultured T cells from kids given the acellular or whole cell vaccine with pertussis proteins (again the same three found in the acelluar vaccine).  The T cells made after whole bacteria vaccine responded by making more cytokines than the ones made in response to the acellular vaccine.  These T cells also divided after detecting the pertussis proteins and were twice as likely to make cytokines and divide at the same time.

These are early studies, but it seems that the T cells may be what differentiate the two vaccines.  None of these basic immunology studies followed kids over time to see whether they became infected.  Hopefully this last study will encourage researchers to look for any relationships between T cell responses and long term pertussis immunity.

Sources:

Differential T- and B-cell responses to pertussis in acellular vaccine-primed versus whole-cell vaccine-primed children 2 years after preschool acellular booster vaccination. SchureRM, et. al. Clin. Vaccin Immunol. Sept, 2013

Impaired long-term maintenance and function of Bordetella pertussis specific B cell memory. Stenger RM, et al. Vaccine. Sept 2010

Different T cell memory in preadolexcents after whole-cell or acellular pertussis vaccination. Smits K, et. al. Vaccine. Oct 2013