Hitting an Invisible Target in TB Vaccine Design

I have a troubled relationship with Twitter. It’s an unredeemable hate sort of thing. I’m generally an inane mix of angry opinion and low self-esteem so, in theory, we’re perfect for each other. I just don’t feel it, though. I had a quick look for online videos in the same vein of the YouTube self-hypnosis that resolutely failed to rid me of my flying (crashing) phobia. Instead, I somehow ended up discovering that my Worldwide Twitter Rank is 56,082,003, which is actually better than I’d expected given that there are over 300 million users. It also turns out that the most prolific Twitter user in the world is @VENETHIS, who tweets mostly about their video game exploits from the look of things. I was selfishly hoping for something more…impactful?

Anyway, this brings me nicely round to the BCG vaccine (I’m up in the top million when it comes to tenuous segueing). BCG is the world’s most prolific vaccine. Unfortunately, it doesn’t always have all that much impact on TB. Some vaccinated people appear to be protected from infection, others…aren’t. So we need a new vaccine. Only, I’ve often heard it said that there is no natural immunity to TB. As in, the immune system appears to lack an anti-TB protocol - the same anti-TB protocol that a vaccine such as BCG is supposed to prime.

Vaccines, on the whole, work by preparing the immune system to meet a future infection with lots and lots of antibodies. This is where TB poses a challenge. The role of antibodies in protecting against TB is controversial and, in the past, has pretty much been ignored. It’s all about the cell-mediated branch of the immune system, cry the immunologists. Our new vaccine needs to stimulate T-cells not antibody production! In reality, of course, it’s far more complicated than one or the other in isolation, so it’s exciting to see recent renewed interest in understanding how antibodies play a role in fighting off TB.

A new PNAS paper from Babak Javid’s lab in China looked at healthcare workers repeatedly exposed to TB who remained healthy. For the uninitiated, not everyone who comes into contact with the pathogen will develop active TB. Around 10% will completely fight off an infection before it takes hold. Of the rest, 90% will succeed in keeping an infection in check. The people who do develop TB are actually a minority, albeit a minority that tallies up to around 10 million cases a year. So understanding what is different about those who control TB compared to those who don’t is a big deal. It can provide important clues to understanding what, exactly, constitutes a protective immune response against TB. Answer this, and vaccine developers would have a real target to aim for.

Javid and colleagues wanted to look more closely at those sidelined antibodies. They picked a busy TB hospital in Beijing where protective measures to stop healthcare workers from being exposed to TB are sorely lacking (that’s an entirely different post), and isolated antibodies from 48 healthy workers and 12 active TB patients. Everyone had anti-TB antibodies, as expected, and there were more in those actively fighting the infection. The big question was if any of these antibodies do anything useful?

So the scientists ‘vaccinated’ mice with the purified antibodies and exposed the mice to TB. Interestingly, antibodies from 7 of 48 healthcare workers provided the mice with moderate protection against infection. It's worth noting that this protection needed the mice to have a functioning cell-mediated immune response which, in the authors’ words, suggests that the protective antibodies “are part of a complex interplay between the pathogen and host immune system.” In short, there’s no one answer to what protects some people against TB and no single cell type holds the solution to designing a better vaccine. Antibodies are likely one piece of the puzzle, but a piece that shouldn't be ignored.

Going back to that old saying about there being no natural immunity to TB - this is based on the observation that patients who've previously had a TB infection are susceptible to catching it all over again. Only it ignores everyone who doesn't develop the disease in the first place. It's like claiming that all toddlers hate broccoli based on a questionnaire posted on a 'my child won't eat vegetables' advice forum. Maybe that anti-TB immune protocol does exist, after all, just not in the people who are generally studied when it comes to TB - i.e. TB patients.

Rebranding Tuberculosis

If infectious diseases were monsters, what would they look like? I imagine malaria would be a terrible mosquito-like creature made of bones, with a wickedly sharp proboscis and a throbbing gut of fiery red blood. Diarrhoeal disease would rise from a swamp of sewage, grinning with its skull’s jaw as it handed out cups of contaminated water to the unsuspecting. And tuberculosis? The biggest infectious disease killer in the world, with 1.8 million deaths from the infection in 2015 and 10.4 million new cases? I struggle to anthropomorphise TB and part of the reason for this is all thanks to the Victorians.

TB was the subject of a 19th century romanticisation that existed in direct opposition to the reality of the disease. Literature, operas, poetry and plays of the day were filled with references to TB, or consumption as it was commonly known, as something to aspire to. In Metzengerstein, Edgar Allan Poe writes: “I would wish all I love to perish of that gentle disease. How glorious! to depart in the hey-day of the young blood - the heart all passion - the imagination all fire - amid the remembrances of happier days - in the fall of the year - and so be buried up forever in the gorgeous autumnal leaves!” Sounds like a blast, doesn’t it?

When I started writing Catching Breath - The Making and Unmaking of Tuberculosis I wanted to cover this romanticisation of TB as an example of how the disease had left its mark on human culture. The poetic swooning! The tragic heroines cut down in the bloom of their lives! The feverous intensity of these martyrs to creativity and passion. Only, I don’t think it is possible to focus on this historical portrayal of TB without adding to the ‘TB is a disease of the past’ story, which I didn’t want to do. Catching Breath was about what has made TB the disease it is today and how we’re going to unmake it. So I left the 19th century romanticisation to other writers and tried to focus on the here and now.

I couldn’t get away from this disease of past thing, though. Whenever I mentioned TB to one of my non-scientist friends, I invariably heard something along the lines of ‘didn’t we already cure it?’. It felt like I was living in two worlds: one in which TB is an underfunded humanitarian disaster the full extent of which we still don’t fully grasp (the TB world); and one in which TB is a throwback to a bygone era that just doesn’t evoke all that much fear or concern compared to something like malaria or HIV, which most know are massive problems in world medicine (the everyone else world).

There’s a lot of great TB advocacy going on that gets across the true toll, but it’s taking its time to worm its way into the public perception of this disease among those living in low TB incidence countries. I think this harms TB control efforts. While TB is not a big problem in high income countries such as the UK, eradicating the disease is not going to happen without partnerships between those countries worst affected and richer countries like my own. And the ‘disease of the past’ stereotype does little to keep the reality of TB at the front of people’s minds.

I don’t think it’s any coincidence that HIV has gone from a death sentence to a manageable condition in just twenty years alongside a brilliant example of how infectious disease advocacy should be done. The HIV world succeeded in branding the infection as a modern monster that could be defeated, just so long as the funding and the political will was there to make it happen. Could we see the same for TB? I don’t know. It’s a difficult task as TB has spent a very long time twisting itself up with human lives. And in the past, the TB world hasn’t done the greatest job of convincing people that a) TB is a worthy opponent and b) it’s a fight we can win. Things are changing, though.

Catching Breath ended up as a scientific biography of TB that touches upon some of the ways in which the disease has shaped the various populations its encountered during its long history, minus the romance. My career as a scientist was all about finding ways to understand the TB bacillus in the hope that we might, one day, be able to truly turn it into a piece of history. For now, though, TB is very much here to stay.

Refocusing the Microscope

I grew up staring out at the stars through my parents’ antique telescopes; marvelling at the tiny pinpricks of twinkling light and how, on a clear night, the Milky Way streaked across the sky. There are more than 100 billion stars in our galaxy, and more than 1,000 billion galaxies in the universe. How many of them, I used to ask myself, contained planets that were home to life like our own sphere of rock and ocean? It was always the potential for life that fascinated me, be it aliens with copper in their blood and sulphurous breath, or plants with red leaves and a taste for nickel. It felt like us humans were just a small part of something infinite in its vastness and, when I thought about it too hard, I became a lone comet tumbling through 46 billion light years of unknowable space.

When I grew tired of feeling small, I played with my parents’ brass microscopes, with their chipped lenses and seized knobs. At first it was leaves and hair and globules of pond water dripped directly onto the mirrors. I never saw very much but the hidden microscopic world fascinated me as much as looking out at the stars. I must have been about ten when the concept of bacteria first took hold of me. I think it was via a book mentioning Anton Van Leeuwenhoek who, back in the 17th century, had fashioned himself a homemade microscope to look at what he described as ‘wee animalcules’ and ‘cavorting beasties’ in fresh water. Of his animalcules, Leeuwenhoek said ‘ten thousand of these living creatures could scarce equal the bulk of a coarse sand grain.’ My view of the universe we live in stretched a little further, much like it had the moment when I’d realised the stars could all be someone else’s sun.

I grew up to become a microbiologist and not an astronomer. From a distance, both fields looked similar to me. Both saw the universe through lenses and mirrors, only one was looking up and the other down. I wanted to see the smallest living creatures in the world because, if we don’t even understand the extremes of life on our own planet, how can we hope to comprehend the breadth of life to be found throughout the rest of existence. The microbial universe was as beautiful as the night sky, with the way Bacillus subtilis formed fractal-like patterns across an agar plate or the rainbow hues of cyanobacteria radiating from the edges of the Yellowstone hot springs. Even the pathogenic species could be wondrous in the way that, wherever you look, life has found a way and a home.

I didn’t intend to work on a pathogenic species at first, but the days of Leeuwenhoek observing his animalcules are long gone. Microbiology, on the whole, is all about using our knowledge of microbes to better humankind—by finding a cure for tuberculosis, in the case of my own research. I envy astrobiologists their timeline, in a way. How they are still explorers who, one day, may be the first to see alien cavorting beasties in a droplet of water from Neptune or the traces of past life in a sample of silica from Mars. But I suppose any field can lose its shine when you zoom in to focus on the minutiae. That’s what happened for me, at least.

From the world’s biggest infectious disease killer, to a yellowy suspension of cells shaking in an incubator, to a fingerprint of proteins making dark bands on a Western blot. Somewhere in my ten-plus years in the lab, the beauty of the microbial world got lost among diagrams of signalling pathways and overly simplified models to distill the behaviour of bacteria down into something predictable. Does it remove some of the wonder of the solar system to understand the physics that hold our planets in orbit around the sun? Only if you spend so much time hunched over your mathematical formulae that you forget to look up at the sky from time to time.

So I left. There were other factors involved in my decision, of course. The birth of my daughter, for one, which re-centred my view of the universe around this one, impossibly bright point of light. But mostly it was that I’d spent too long looking down that microscope and had lost sight of everything else. The day after I handed in my resignation, a publisher at Bloomsbury messaged me to ask if I’d ever thought of writing a popular science book. Two years later, Catching Breath - The Making and Unmaking of Tuberculosis is on its way to publication. I started out intending to shine a light on how Mycobacterium tuberculosis, the bacterium behind the disease, is the cleverest of them all. Only, the gravitational pull of my own cavorting beastie kept changing my focus. It’s no longer M. tuberculosis that fascinates me, but the place it occupies in the world.

I used to think of the microbial universe as something separate from us, to be observed from a distance. Only, microbes—the ones on our skin, in our guts, floating around in our drinking water, or causing infections such as TB—make up the fabric of our lives. We exist in a vast, interconnected world in which no one species lives its life in isolation. There’s no good guys and bad guys here, not really; just a huge tapestry of life that isn’t always sewn in humankind’s favour. Catching Breath isn’t a zoomed in review of the scientific research into M. tuberculosis. I’ve taken a step away from my own work and tried to tell a story of how TB fits into the rest of the world and, in doing so, I’ve remembered why I got into research in the first place.

When Bad Bugs Go Badder

The Beijing lineage of M. tuberculosis is the villain in a movie sequel. Nastier, scarier, harder to kill. You thought tuberculosis (TB) was bad? Think again. The Beijing lineage is that little bit worse, associated with a speedier disease progression and increased antibiotic resistance.

I’ve always had a thing for studies that attempt to pick apart the origins of infectious diseases. So when I spotted a paper on the makings of the Beijing lineage, my excitement levels came close to when I discovered there’s going to be a Guardians of the Galaxy 2.

A while back, a paper came out suggesting that the Beijing branch of the TB family tree emerged some 30,000 years ago in Southeast Asia. Then, as the Han Chinese population embraced farming as a way of life, BOOM! The Beijing lineage exploded in northern China and started its journey towards worldwide misery.

Another study, however, put the approximate age of the lineage at around 6,600 years old. Suffice to say, attempts to date the M. tuberculosis family tree are notoriously unreliable. They all rely on some pretty big—and pretty shaky—assumptions, mainly based around how quickly the bacterium accumulates changes in its DNA.

So on to this new piece of research. It takes advantage of the increasing availability of whole-genome sequencing to look at how the pathogen has evolved in fine detail. The researchers were interested in a specific branch of the Beijing lineage—the central Asian clade, which recently reared its evil little head in Western Europe.

Like previous studies, I doubt that the molecular clock used in this study was calibrated accurately. But what’s clever about work like this is that it’s possible to superimpose expansions in the bacterial population size over big human upheavals. More bacterial diversity=worse living human living conditions. TB basically takes advantage of low points in human history. Anything that results in poor health, overcrowding, decaying healthcare systems, etc etc.

The study suggests that back in the 1950s to 1960s, the central Asian clade first appeared in the former Soviet Union. Then, thanks to the Soviet-Afghan war, it entered Afghanistan 1979-1989. Following the American invasion in 2001, the clade spread further as people were displaced from their homes and former lives. As Afghan refugees found their way to Europe, so did the central Asian clade of the Beijing lineage.

Back in the former Soviet republics, the clade had been similarly busy taking advantage of the fall of the Soviet Union. When it comes to historical events with huge ramifications for TB, the implosion of the Soviet Union is among the big ones. Bye bye, decent public healthcare and TB control; hello, drug resistant TB. Even today, multi-drug resistant TB is a gigantic issue in many ex-Soviet countries.

What studies such as this one demonstrate is how easily M. tuberculosis rides upon the back of political instability, war, population displacements and all the other crap us humans heap upon each other. And based on current events, none of that crap is going anywhere. So where does that leave TB?

Eldholm V, et al. (2016) Armed conflict and population displacement as drivers of the evolution and dispersal of Mycobacterium tuberculosis. PNAS 113(48):13881-13886.

The Missing Three Million

In Agra’s slums, community volunteers are visiting the houses one-by-one and asking the occupants a simple question: “Have you been coughing for more than two weeks?”

Of the ten million new cases of TB every year, one-third remain invisible to the public health authorities. India currently holds the dubious title of World’s TB Capital and accounts for one million of those missing patients. Many of those whose TB goes either undiagnosed or unreported are lost somewhere among the muddy streets and flapping fabrics of the poorest urban communities.

Much of India’s TB problem is socioeconomic—poor housing, poor sanitation, overcrowding, and unhealthy populations both at higher risk of developing active TB and with limited access to adequate healthcare. Active case finding among these marginalised and vulnerable populations is part of the solution to ensuring that people receive treatment and do not continue to transmit their infection to others.

A short paper published last week describes the lessons learnt from a pilot project looking for active TB cases among the half-a-million inhabitants of ‘Agra city’. Community volunteers were offered incentives to visit house-to-house and educate the inhabitants on TB, collect demographic details, and ask if anyone had been coughing.

Where the answer was ‘Yes’, potential TB patients were referred to a local health facility for further testing. What surprised me about the results of the study was that only 40% of those referred actually went of their own fruition. The other 60% had to be accompanied by the community volunteers, who returned to the homes of those not self-presenting within one week.

The study also revealed that levels of TB knowledge among the 3,940 households surveyed were actually very high. Ninety-percent of respondents had heard of TB; most of these people knew that coughing was a symptom. Yet the volunteers still managed to find 382 potential TB patients who hadn't sought out a diagnosis.

When questioned, most of the families said they relied on private healthcare providers for medical care. India’s private sector is highly variable, encompassing world-renown TB doctors all the way down to unqualified charlatans. The combination of patients not seeking out medical care and, when they do, turning to someone who won’t necessarily provide them with a correct diagnosis begins to explain why so many cases of TB go undetected.

It was beyond the paper’s scope to discuss why patients don’t seek out a TB diagnosis but, for me, this is one of the most important questions. What are the barriers that stop people living in Agra city—or in any other part of the world, for that matter—from approaching the health services with symptoms of TB?

Because new drugs, vaccines and diagnostics aren’t going to eradicate TB alone; not when millions still don’t receive the existing treatments and people continue to die from what is a curable disease.

Prasad et al. (2016) Lessons learnt from active tuberculosis case finding in an urban slum setting of Agra city, India. Indian J Tuberc. 63(3):119-202

Blame the Europeans

You know when you drive an unfamiliar car and you have to find your way round all these knobs and buttons to make the car go in the direction you want it to go in? M. tuberculosis has the same problem when it comes to the human immune system. This can make things tricky as it’s a pathogen that practices immune subversion rather than immune evasion—driving the immune response in its own favour rather than hiding.

A lot of the time, specific lineages of TB stick to the human genetic backgrounds that they’ve grown-up infecting. Even as global travel increases and the world gets smaller, these associations between TB lineage and their preferred flavour of human host persist. Over millennia, specific lineages of M. tuberculosis have evolved alongside specific human populations, learning all our secrets and finding ways to navigate our immune systems. Faced with an unfamiliar host, however, the pathogen can struggle.

But some lineages and sub-lineages are better at adapting to new host backgrounds than others. They’re the ‘generalists’ of the TB family. In comparison, the less-adaptable strains are known as ‘specialists’ and more or less stick to geographically restricted populations. They’re the ones who, if they ever go on holiday, demand a hire car exactly like the one they have at home. ‘I can’t be dealing with these fancy Japanese cars’, they say. ‘Give me a nice, British-made Rover.’

This idea of co-evolution and adaption is one I talk about in more detail in my book Catching Breath - The Making and Unmaking of Tuberculosis. The book is basically a scientific biography of TB exploring how M. tuberculosis came to be the world’s biggest infectious disease killer and how science is going to kill it right back. It comes out next summer as part of the Bloomsbury Sigma imprint of popular science books.

Here, though, I wanted to mention a new paper that came out last week from Sebastian Gagneux’s lab in Switzerland. In it, he looks at the success of M. tuberculosis lineage 4. It’s the most adaptable lineage, making its home on every inhabited continent—a generalist lineage that’s found its success thanks to both biological and social phenomena, according to Sebastian’s study. 

The team used SNP-typing and targeted whole genome sequencing to look at the genetic differences between 3,366 lineage 4 strains isolated from 100 different countries. They used the differences to break down lineage 4 into several sub-lineages. Some were generalists found in multiple locations; others, in comparison, were more isolated and rarely strayed outside of small regions of Africa, for example.

Like I said, M. tuberculosis doesn’t hide from the immune system. It wants to be recognised so the bits of the pathogen on the immune system’s watch list don’t tend to vary much at all. But Sebastian’s study showed that the generalists are more immunologically versatile than their specialist counterparts. This is likely because they have evolved to deal with a wider range of host genetic backgrounds and have had to come up with ways to ensure they get their own way no matter who they infect.

The paper was also interested in the sub-lineage known as L4.1/LAM. L4.1/LAM, like Starbucks, is on a mission to take over the entire world. It’s found everywhere from Africa to Australia. The scientists used the genetic differences between members of L4.1/LAM to predict its geographical origin. According to the results, it first emerged in Europe then, as us troublesome Europeans spread around the world, we took our TB with us.

It’s a common story when it comes to TB. The disease is among the world’s oldest and its history is twisted up with that of us humans. The spread of TB around the world tracks with human migrations; explosions in M. tuberculosis populations overlay with human upheavals. Understanding the co-evolution of M. tuberculosis and the human immune system has consequences for, in particular, vaccine design. How do we vaccinate against a pathogen that uses the immune response for its own benefit and how do we make sure any vaccine works in every population where not only the predominant lineage differs but so does the host’s genetic makeup?

Stucki D et al. 2016. Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sub-lineages. Nature Genetics.

Yu Y et al. 2015. RASP (Reconstruct Ancestral State in Phylogenies): a tool for historical biogeography. Molecular Phylogenetics and Evolution. 87: 46-49

Wasted Time Isn’t Always Wasted Time

The other day I commented to the father of my child that having a baby is a little bit like going to prison. Not the ‘nice’ sort of prison where they let you do Open University courses and try to make you a better person. A Victorian-style prison where the inmates are forced to turn a crank thousands of times a day or walk on a treadmill for hours and hours with no end product to show for their labours. You do your time and then you’re released into a world you no longer recognise, having lost half your hair and a year of your life.

You know, writing that makes me realise why my partner looked at me with his ‘What the fuck?’ face. Yes, yes, I have a one year old miniature human to show for all those hours spent pacing to and fro sob-singing Somewhere over the Rainbow while the baby screamed because she was so flipping tired that she just couldn’t sleep. And, yes, all that panic-Googling because said baby has a highly disturbing habit of rolling her eyes over white when it’s windy wasn’t entirely wasted. I now know more about neurological disorders in infants than the average neurologist.

But I can’t say the first year of my baby’s life has lived up to my expectations of motherhood. Don’t get me wrong—it’s been totally worth it. My child really is the best baby in the world, even taking into account that month-long phase where she got so fat that all our baby photos look like we’ve dressed a small pig in human clothes. Or that way she hisses and bares her teeth whenever there’s a bright light nearby, like some blood-sucking vampire grub. Or the time she gave me Hand, Foot and Mouth disease that adults totally aren’t meant to get, which I totally did get, and am in fact currently typing with a fingernail that is totally going to fall off any day now, and urggggghhh.

I know what you’re thinking: if this is Kathryn’s first foray into Mummy-blogging then, Dear God, nooooo. You’re alright, don’t worry. I am fully aware that, while I’ve somehow managed to find myself in possession of a predominantly happy and healthy toddler, it has not been thanks to any previously dormant Mother-skills that need to be unleashed upon the world. I barely know what I am doing when it comes to my own child, so I am most definitely not in any position to offer parenting advice to others.

What I do want to talk about is how wasted time isn’t always wasted time. I worked in scientific research for, what, 8 years as a post-doc and for 12 years total if you count the time spent working towards my PhD. And of all the work I did during that time, maybe 5% amounted to something useful. Maybe less, depending on your feelings towards basic tuberculosis microbiology. All those hours in the lab, all that funders' money. You can’t be scared of failure if you want to be a scientist. 

I’ve read papers before where years of work have been condensed down into a few lines. The protein could not be crystallised. A gene deletion mutant showed no phenotype. Compounds showed poor activity in vivo. No one gets into science to spend their days performing the grunt work that provides the filler for the rare discovery that changes the status quo. But the reality of working in the lab is that taking the easy road rarely leads to the really interesting results, but taking a risk—trying something completely new—will often lead absolutely nowhere.

Now that I’ve left the lab and am embarking on a new adventure in the form of writing a popular science book, I am getting to see science from a new perspective. It’s dizzying to see how much research there is out there that no one outside the field ever hears about. Work that’s published in the best journals can still be just another drop in the ocean. Years, sometimes decades, of work. Even the biggest discoveries can sometimes look very boring from the outside. It’s enough to make my own work feel very small and insignificant.

But somehow, while you’re hunched over the lab bench, it doesn’t feel like you’re wasting your time. Two weeks struggling to make a protein expression vector. Two months purifying a protein so that you can get started on the real experiments. Two years screening inhibitors only to conclude that the protein you picked at the start wasn’t the best drug target after all. Go back to the beginning and try again. From the outside, it looks like wasted effort but, along the way, there were small successes. New techniques that will improve future attempts. Students trained who will go on to do their own research. Interesting side projects that may or may not lead to something exciting.

I’d rather hoped that the lessons science taught me about the value of failure might translate into a bottomless well of patience when it came to stay-at-home-parenting. Hypothesis rejected! One of the issues is that reproducibility goes out the window when it comes to babies and there’s an inverse relationship between how much research you do and how good the outcome is. But, like my scientific career, I sometimes look back on the first year of my baby’s life and wonder where all the time went. What have I achieved? Could I have done more?

Now that I’m coming out the other side of the baby period and starting to piece back together my own life and ambitions, I can see why so many women find it so difficult to juggle motherhood and a career. Pre-baby, it seemed so simple. I couldn’t understand why some women seemed to cease to exist as an individual once they had kids. Now? I can’t decide whether those failures in the lab would feel like time that could have been better wasted at home with my daughter, or vice versa.

So instead of returning to work for someone else, I am going to attempt to carve out a freelance writing career that will let me work on something I want to work on while also being around to clean spaghetti off the walls. No doubt there will be plenty of failures along the way and I am sure there will be times where I regret wasting my time on something that leads nowhere. But, hopefully, in a year’s time when I look back, it will have all have been worth it.