ImmYOUnology

More than just vaccines


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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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Update on antibiotic resistance week: FDA makes recommendations to limit antibiotics in agriculture

I mentioned in my post on antibiotic resistance that most farm animals in the U.S. are constantly on antibiotics to prevent disease and promote growth (these two outcomes are not necessarily linked).  In the E.U. this practice was halted years ago to avoid the risk of antibiotic resistance from developing.  It seems that the U.S. F.D.A. is also going in this direction. They put out a policy that recommends suspending the use of antibiotics for healthy animals. Check out these two links to learn more:

http://www.nytimes.com/2013/12/12/health/fda-to-phase-out-use-of-some-antibiotics-in-animals-raised-for-meat.html?smid=fb-share&_r=1&

 

http://www.wired.com/wiredscience/2013/12/fda-213-vfd/#more-390021


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I missed antibiotic resistance awareness week!

Last week, Europe, Australia and the U.S. all launched their 5th Antibiotic Awareness Weeks, spreading the word about bacterial resistance to antibiotics. I heard of the event during a conversation about the death of a New Zealand man who had been infected with pan-antibiotic resistant bacteria.  According to reports, the infection was not the cause of his death, but it made news because his doctors found that no known antibiotic could stop his infection.

On her blog, Superbug, writer Maryn McKenna closely follows the growing issue of antibiotic resistance. She also recently wrote a report about what the world might look like without any working antibiotics. Recall any time you’ve taken antibiotics for an ear infection or after a root canal or a simple surgery. Those simple infections and procedures could become a lot more dangerous without the ability to kill off bacteria.  After all, the first test case for penicillin was a gardener who was dying of an infection caused by a small cut from a thorn.

A very well illustrated CDC report estimated that 2 million people are infected with antibiotic resistant bacteria in the U.S. each year.  In an effort to bring some organization to the overwhelming issue, the report ranked 18 resistant organisms as “urgent,” “concerning” or “serious” threats.

What does resistance mean exactly? And what does this have to do with immunology?

Our immune systems have a variety of techniques to address bacterial infections. We have cells called phagocytes that eat bacteria and digest them, we have specialized T cells, natural killer cells and neutrophils that secrete molecules and proteins that damage bacteria or kill infected cells. Proteins in our blood (complement proteins) stick to bacterial cell surfaces and then build upon each other like blocks until they make what’s called an “attack complex” that drills a hole in the bacterial cell membrane. 

{Did you know we need free radicals to fight infections and kill cancer cells?  Phagocytes use them to kill the bacteria that they engulf.  Many people think of free radicals as “bad” molecules and take antioxidants hoping to prevent cancer, but free radicals are one way our bodies destroy rebellious cancer cells. In fact, many cancer treatments activate free radicals to target cancer cells and patients receiving these treatments are instructed to avoid antioxidant supplements.}

If a species of bacteria is good at avoiding one tactic, there are plenty of others, and a healthy immune system will usually contain an infection and eliminate it. If someone has an unhealthy immune system (immunocompromised) or the inflammation from the immune response starts to damage tissue, antibiotics are a great way give the immune cells a fighting chance and spare the infected person.

Unlike the immune system, an antibiotic is just one compound with one tactic or mechanism. So it’s possible for just one mutation to give a bacterium resistance to that one compound’s tactic. If a drug targets a protein needed for bacterial cell membrane construction, the bacteria may alter the protein, change its construction method or make a different protein that pumps the drug out of the cell.

DNA mutations are common at the rate that bacteria divide.  Staphylococcus aureus, for example, can divide every half hour. I grow S. aureus in the lab, and when I put an amount that could fit on a pen point into a tube of special broth at 37° C (the same temperature as the human body), the next day I end up with enough bacteria to fill about a quarter of a teaspoon.

Life for bacteria is tougher inside a human body since they are always being attacked by the immune system. But say the bacteria get the upper hand inside an infected person, we’ll call her Amy. Amy goes to the doctor and gets an antibiotic prescription, perhaps methicillin or vancomycin—enough pills to take one a day for 7 days. When she starts taking them many bacteria will die or stop dividing and her immune cells will be able to make headway against the infection.  S. aureus can accumulate hundreds of mutations a day and even though most of the mutants will die, there will likely be individual bacterium inside of Amy with mutations that make them resistant to the drug she is taking. Those resistant mutants have an advantage over the other bacteria and will take over as all of their siblings get killed off by the drug.

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Imagine the image shown on a kitchen cleaner commercial as it touts “kills 99.9% of germs.” On one side of the screen the “before” shot shows lots of wiggly cartoon “germs” and on the other side, after the cleaner is used, there are one or two wiggly little guys left.  By “99.9%,” the commercial means that their product kills most known household species of bacteria, but if those were all the same species infecting a person and the cleaner was an antibiotic drug, those two little guys left behind would multiply with a vengeance and birth a resistant strain.  There is a potential for that to happen every time a person takes antibiotics.

Ideally, even if mutations do arise, the antibiotics slow down the bacteria enough to let the immune system completely eradicate the infection (including the resistant mutants).  But say Amy takes her prescription for 4 days and feels like she’s back to normal so she decides not to finish the whole 7 day course. By this time, she may be harboring resistant mutants and when she stops taking the drug, her immune system will suddenly have a lot more to deal with, and the mutants may expand.  When she feels sick again a day or two later and starts to take the antibiotics again, they may not work at all since the resistant mutants have outgrown the other bacteria. She may end up having to get a different antibiotic injected into her bloodstream in the hospital and while she’s there, she may spread the resistant bacteria to other patients.

That’s the story of one mutation giving rise to a bacterial strain resistant against one drug.  The man who passed away in New Zealand was infected with a strain of Klebsiella pneumonia that was resistant against every drug. How did that happen?

The short answer is we don’t know the history of every resistant strain. One interesting example that scientists are following is vancomycin-resistant S. aureus (VRSA). VRSA has been isolated about a dozen times from individual patients.  Most of them were diabetic patients with methicillin-resistant S. aureus (MRSA) infections that coincided with the presence of vancomycin-resistant Enterococcus sp. (VRE).  It’s thought that the VRE transferred its genes for vancomycin resistance to the MRSA, resulting in a strain of S. aureus that is resistant to both methicillin and vancomycin.  This sort of gene transfer can happen inside an infected person or out in the environment.

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Scientists are also monitoring how the use of antibiotics in agriculture and aquaculture may promote development of antibiotic resistance.  According to a 2010 review, less than half of all antibiotics are used to treat human infections. Most farm animals are constantly on antibiotics and are known to harbor resistant strains of bacteria, making foodborne illness a potential route to spread resistant bacteria or genes to humans.  To microbiologists, chronic use of antibiotics in agriculture is an obvious route to resistance, but it’s difficult to track exactly where resistant strains originate, so the issue is still controversial.  A study published a few days ago found that MRSA infection rates were statistically correlated with how close Pennsylvania residents lived to livestock feedlots or to fields where swine manure was used as fertilizer.

There is a large gap in our knowledge about the link between antibiotics and agriculture, but it is an active area of research.  Whether you buy antibiotic-free meat or whether you cover your house with Lysol, the one non-controversial and vital thing we can all do is always complete the full course of an antibiotic prescription.

Sources

NZs first ‘superbug’ victim? Siouxsi Wiles Nov 2013

The real story behind penicillin. Howard Markel, MD Sept 2013

Antibiotic resistance threats in the United States. The CDC, November 2013

Antibiotic resistance, mutation rates and MRSA. Pray, L. Nature Education, 2008

The emergence of vancomycin-intermediate and vancomycin-resistant Staphylococcus aureus. Appelbaum, P. Clin Micr and Inf Jan 2006

The Origin and Evolution of Antibiotic Resistance. Davies, J. and Davies, D. Microbiol Mol Biol Rev Sept 2010

A call for antibiotics alternatives research. Stanton, T. Trends in Microbiology March 2013

MRSA: Farming up trouble. Mole, B.  Nature July 2013

High-density livestock operations, crop field application of manure, and risk of community-associated methicillin-resistant Staphylococcus aureus infection in Pennsylvania. Casey, JA et. al. JAMA Intern Med Nov 2013


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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


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Could whooping cough make another comeback this winter?

Signs outside of Walgreens and CVS have been advertising the flu vaccine for several weeks now.  Even if its effectiveness varies from year to year, I consider it well worth the shot since I work around undergraduates and hospital personnel.  Something I don’t expect to see this winter are advertisements for DTaP (diphtheria, tetanus, acellular pertussis) boosters even though they may be just as important for some as flu shots.

Pertussis, also called whooping cough, is caused by a lung infection with the bacterium Bordetella pertussis. It may sound like a nineteenth century disease you’d catch along the Oregon Trail, but whooping cough is a modern issue and can be serious or fatal, especially for newborns. According to Centers for Disease Control, the last few years have brought the largest pertussis outbreak since the 1950’s, reaching over 50,000 cases in 2012 (compared to a low of about 1000 in 1976).  So, if we have a vaccine, why are there outbreaks?

In the 1950’s widespread use of the DTP vaccine (diphtheria, tetanus, pertussis) began in the U.S. In a couple of decades, the number of whooping cough cases dropped from about 50,000 to fewer than 1000.  The vaccine was effective, but in the late 1970’s and then the 1980’s the vaccine’s side effects took center stage, perhaps as the memory of the disease faded.  The DTP vaccine caused some combination of redness, swelling, pain and fever in about half of the children vaccinated.  Some more serious reactions, like seizures were reported, but were transiently caused by fever and never led to permanent problems.  Concerns that the vaccine caused neurological damage could not be substantiated and throughout the 1980’s, scientists reported that the risk of getting whooping cough outweighed the cost of the side effects.

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(http://www.cdc.gov/pertussis/surv-reporting.html)

From our point of view, it may seem brutal to accept such risks, but at the time, DTP was the only option available to prevent a disease that could be much more devastating.  Whooping cough is named after the sound that infants make when they try (sometimes unsuccessfully) to take a breath in between coughing fits.  The infection destroys structures in our lungs called cilia, which are tiny protrusions that collectively brush out grime from our lungs each day.  They are like people in a mosh pit passing unwanted particulates out the door of your airway.  When cilia are disabled, mucus collects in the lungs and your body copes by inducing spastic, uncontrollable coughs.  Some can be extreme enough to slip vertebral discs or break ribs.  The cough can last for months.  For infants, the results are much worse because their lungs and airways are small so they have a much harder time catching their breath.  This story gives an idea of how helpless parents and doctors can be to help an infant with pertussis.

Back to the 1980’s.  Once the fear of the vaccine overcame fear of the disease, a new option had to be explored.  The DTP contained whole bacteria that could not cause infection, but was causing inflammation in many vaccine recipients.  Inflammation occurs when different types of immune cells gather in large numbers and release proteins that expand blood vessels and recruit more immune cells. Swelling follows and the cells release compounds that are meant to damage bacteria, but can also damage tissue.   The process is usually well controlled and only lasts as long as it takes to remove whatever started it all off.  The DTP vaccine, it turned out, caused inflammation because of a type of molecule called endotoxin in bacterial membranes.  Endoxin non-specifically binds and activates immune cells and causes unchecked inflammation.  So work began on a form of the vaccine with individual purified pieces of B. pertussis, called an acellular vaccine. It was approved as a booster by 1991 and the DTaP (diphtheria, tetanus and acellular pertussis) replaced the DTP completely by 1996.

It took about a decade to see the pattern emerge, but it’s starting to become clear that immunity after DTaP immunization does not last as long as immunity conferred by DTP.  Since the vaccine requires 5 boosts, one potential cause could be under-immunization, or a failure to complete the whole vaccine course.  A 2010 study reported that California kids who tested positive for pertussis were less likely to have received a fifth booster, which suggested that a full course was important for protection.  But in the same study, many kids who did get the fifth boost were found in the infected group.   These kids were also more likely to have received the last boost over a year prior to the study. That meant that even after five doses, the vaccine seems to wear off after a year.  A more recent study done in Minnesota and Oregon also showed that kids’ susceptibility to getting pertussis increased a little more each year after the last booster.

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(http://www.cdc.gov/pertussis/surv-reporting.html)

So, under-immunization is not the whole story and although the DTaP vaccine works for about a year, there is something fundamentally different between it and DTP.  It’s been suggested that it doesn’t cause enough inflammation or not the right kind of inflammation. So far, there is not enough basic science to support these ideas so researchers are taking a step back to ask simple questions about how the vaccine actually works (More on this in my next post).

Another idea is that B. pertussis is mutating and since DTaP only has a few of the bacteria’s proteins in it, dividing bacteria can start producing fewer or none of those proteins.  This possibility is driving some scientists to explore different vaccine designs with more diverse proteins or to go back to a whole bacteria vaccine (like DTP), but without the super-inflammatory endotoxin.  These explorations will depend on funding and it will take a long time to show that any new vaccine is effective, safe and cost-effective.

In the meantime, parents and doctors have to decide how best to use the existing vaccine to protect kids from whooping cough.  Infants are the most vulnerable to the disease but they can’t be vaccinated for a couple of months after birth.  Doctors and public health officials have turned to boosters for adolescents and adults with the idea that if parents and siblings are protected, they will be less likely to pass the infection to a newborn.

In 2005, the CDC recommended a 6th booster called Tdap for adolescents between 10 and 18 years old.  One study tested the effectives of this method by comparing observed infection rates among infants to rates that were estimated based on data from previous years. They found that actual rates were significantly lower than the projected ones, bringing hope that cocooning may work.  This year, Tdap boosting during pregnancy was deemed safe and was recommended by the CDC.  Hopefully another year or two will bring evidence that vaccinating moms during each pregnancy reduces infant pertussis.

While researchers work on finding a happy medium between the inflammatory whole bacteria DTP and the fair-weather DTaP, we are left with rudimentary options: get Tdap boosters, keep kids up to date on their DTaP doses and wash your hands long enough to sing the “happy birthday” song three times.

Sources

“The Pertussis Paradox” Allen, A.  Science Aug 2013

The Immunization Action Coalition

The CDC

Nature and rates of adverse reactions associated with DTP and DT immunizations in infants and children. Cody, CL.et al. Pediatrics Nov 1981

Severe reactions associated with diphtheria-tetanus-pertussis vaccine: detailed study of children with seizures, hypotonic-hyporesponsive episodes, high fevers, and persistent crying. Blumberg, DA et al. Pediatrics Jun 1993

“Pertussis Vaccine: Myths and Realities” Gold, R. Can Fam Physician May 1988

Pertussis and pertussis vaccine: further analysis of benefits, risks and costs. Hinman, AR and Koplan, JP. Dev Biol Stand 1985

Association of childhood pertussis with receipt of 5 doses of pertussis vaccine by time since last vaccine does, California, 2010. Misegades, LK et al. JAMA Nov 2012.

Waning immunity to pertussis following 5 doses of DTaP. Tartof, SY et al. Pediatrics Apr 2013

Tetanus, diphtheria, acellular pertussis vaccine during pregnancy: pregnancy and infant health outcomes. Shakib, JH et al. J Pediatrics Nov 2013.