Tuning down the immune system could improve the flu vaccine

This post is based on an article I recently wrote for an internship application, so it’s more formal than a typical post, but I think it’s a cool story that helps explain how the flu vaccine works. Enjoy! And stay healthy!

After more than 2,000 confirmed cases and over twenty deaths, the 2013-14 flu season is still approaching its peak.  Vaccination remains the best prevention despite the flu vaccine’s hit-or-miss reputation.   Each year the U.S. Food and Drug Administration recommends three strains of influenza that the World Health Organization believes are worth targeting, and six months later the season’s new vaccine is distributed.

This nasty viral particle is trying to get inside a cell. It’s covered in NA (red) and HA (blue) proteins.

One of the flu vaccine’s biggest problems is its inability to induce immunity against multiple viral subtypes.  Subtypes of the influenza A virus, like H1N1 or H5N1 are distinguished by the surface proteins hemagglutinin (HA) and neuraminidase (NA).  The vaccine can protect against a few subtypes at a time, but if a subtype not included in the shot makes a strong appearance one season, not much can be done to prevent it from spreading.  This year, the vaccine is pretty spot on. It includes H1N1 which has been making a comeback this year.

This problem has driven researchers to pursue a universal vaccine that could protect against multiple subtypes.  This type of protection is called heterotypic immunity.  One group of scientists from St. Jude Children’s Research Hospital hit on an unexpected way to expand the reach of one flu vaccine to multiple subtypes.  Dr. Maureen McGargill and her group published their study in Nature Immunology in December.  They studied how a common immunosuppressive drug called rapamycin influenced the ability of vaccinated mice to generate heterotypic immunity.  They vaccinated mice with one viral subtype and infected them with three other lethal subtypes.  Surprisingly, the mice who got rapamycin were better able to resist infection by all the subtypes, including an altered H5N1 strain, commonly known as the avian flu.

Rapamycin is commonly used to dampen the immune system to prevent organ transplant rejection.  It blocks an immune system regulating protein called mTOR.  Three other animal vaccine studies previously found that rapamycin enhanced generation of memory T cells, cells that can remember a virus and kill infected cells when they detect viral proteins.  None of these studies linked higher numbers of memory T cells to protection from infection.  McGargill’s group observed both higher memory T cell numbers and better protection, but could not link the two. Rather, they found that protection was related to changes in the kinds of antibodies that the vaccine induced.

The flu vaccine contains pieces of viral proteins called antigens and mice and humans make antibodies that specifically bind these antigens on the viruses and neutralize them.  The more specific the antibodies are though, the more they drive those proteins to mutate so the virus can escape detection.  This shape-shifting tactic is called antigenic drift, and it is part of the reason it is so difficult to predict which vaccine formulation will be most effective each year.    

The coveted universal vaccine would induce antibodies that recognize parts of the virus that are shared, or conserved, by many subtypes and unlikely to mutate.  But B cells, the cells that make antibodies, tend to make more and more specific antibodies over time.  Over several weeks, B cells go from making weak, broadly binding antibodies that can cross-react with many subtypes, to strong and specific ones.   McGargill and her colleagues found that rapamycin interrupted this process and caused the mice to make more of the broadly binding antibodies.  The antibodies also targeted different parts of the hemagglutinin protein.

The group could not determine exactly how the altered antibodies contributed to protection from infection.  They concluded that the antibodies produced after rapamycin treatment were less specific and therefore able to cross-react with several viral subtypes.   As a result, the treated mice were less susceptible to the three different influenza subtypes.

These findings could be useful for quickly designing broadly protective vaccines in the face of a new subtype outbreak or epidemic.  It currently takes about six months to manufacture the annually recommended formulation.  A heterotypic vaccine would not be as dependent on the World Health Organization’s laborious surveillance and data analysis, and could be stored and used for many flu seasons.

Sources:

Bridges CB et al. Effectiveness and cost-benefit of influenza vaccinations on healthy working adults: A randomized controlled trial. JAMA (2000) 284:1655-63.

Keating R. et al. The kinase mTOR modulates the antibody response to provide cross-protective immunity to lethal infection from influenza virus.  Nature Immunology (2013) 14:1266-76.

McMichael A and Haynes B. Influenza vaccines: mTOR inhibition surprisingly leads to protection. Nature Immunology (2013) 14:1205-07.

Pica N and Palese P. Toward a universal influenza virus vaccine: Prospects and challenges. Ann. Rev. Med. (2013) 64:189-202.

http://www.cdc.gov/flu

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.

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.

*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: 10.1371/journal.ppat.1003592.s001

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: 10.1016/j.jcv.2012.11.011

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.

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.

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

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