The conundrum of influenza A virus diversity and host immune responses – lessons from a vaccination experiment in Mallards

The influenza A virus is in an interesting virus. It exists in many subtypes and can infect a range of hosts, but most of the variation in subtypes and lineages is restricted to wild waterfowl, especially dabbling ducks. Contrary to humans and other mammals, the virus doesn’t normally cause disease in ducks and these viruses are said to be low-pathogenic. The traditional explanation for the evolution of subtypes is that they have evolved to be sufficiently antigenically different that infection with one subtype does not incur protection to another one. Hence, antibodies raised against a H1 virus would do poorly with an H7 infection, and vice versa, but work well against an infection with a homologous virus, i.e. another H1 virus.

The latter is called homosubtypic immunity, and has been shown in a range of studies of Mallards (our favorite bird), using both experimental infections and studies conducted in the field, and although serum antibodies in Mallards seem to wane with time, immunity does seem to be long-lasting (see for instance Tolf et al. 2013).

A few years ago, we identified the existence of heterosubtypic immunity in wild Mallards. We analyzed infection histories of individuals recaptured during their stopover stay at Ottenby and investigated patterns of subtype occurrence compared to what would be if infection order was non-structured. In essence, what we could see was that heterosubtypic immunity was frequent, most strongly observed at hemagglutinin (HA) clade level, but also detectable at the HA group level. In contrast, there was no effect of the neuraminidase subtype (see Latorre-Margalef et al. 2013). The strength of this pattern was rather surprising, and has sparked follow-up studies.

Lately, a number of studies have used experimental infections to investigate heterosubtypic immunity further, either as a cause of understanding how highly-pathogenic viruses can be maintained in waterfowl, or for assessing immunity patterns in low-pathogenic avian influenza infections. Two nice, recent articles are by Segovia et al. 2017 investigating H3N8, H4N6, H10N7 and H14N5 infections in a balanced design, and by Latorre-Margalef et al. 2017 assessing protection of H3 antibodies against a range of other virus subtypes. Collectively, these studies suggest that the order of infections are important for future disease dynamics, both at the individual level but also at the population level. In other words: the order of outbreaks in a population will govern the fate of other subtypes in the population later; a competition among subtypes over susceptible hosts. This is very interesting, and something we currently try to model with infection history data of captured and recaptured wild Mallards at our study site.

The principle of immunity is that previous infections will render the bird immunity to reinfection with the same virus subtype, so called homosubtypic immunity, as long as the antigenic properties of the two strains are similar. A heterosubtypic immunity is when infection with one subtype provides full or partial protection against other subtypes, and it is expected that this is more common in phylogenetically related subtypes. (Illustration by M. Wille)

However, field and lab are two different things, and a couple of years ago we wanted to use the duck trap at Ottenby to study immune processes. As we cannot infect and release birds in the trap we used vaccination as a means of simulating previous infection. We prepared two vaccines, one against H3 and one against H6 (and one sham), immunized birds and followed them to make sure they developed serum antibodies (against NP) and neutralizing antibodies against the HA, after which we released them into the duck trap and followed their natural infections in the wild. As often is the case, our experiment didn’t really go as intended. First of all, there were no H6 infections in the wild population at the time of the experiment, thus no H6 infections recorded in any of the groups of our experiment so we couldn’t analyze the protectiveness of H6 vaccination. Quite surprisingly, all three groups were infected with H3 viruses – including the group that had received the H3 vaccine.

There are two possible explanations for the failed homosubtypic response. One is that immunization didn’t result in protective immunity, and the other that the viruses were antigenically different. We did detect neutralizing antibodies against H3 viruses in the ducks, suggesting these ducks did raise a specific immune response against the vaccine. Interestingly, the ducks didn’t raise a similar response against H3 infections after being in the duck trap. Investigating the latter we could show that the vaccine strain and the outbreak strain differed by a number of substitutions close to the receptor binding site. Going back to our virus neutrilizations, we could see differences in in the strength of the antibody response against different H3 viruses, including differences between the strain we used to vaccinate and the strain that was circulating during our experiment. Sufficiently different to suggest antigenic difference. The paper is just out (Wille et al. 2017). H3s are quite interesting, as they have been the focus in much of human infection research, especially because there seems to be two antigentically different lineages and after infection with one of these H3 lineages humans may not be protected against the other. Antigenic cartography has identified the importance of a few sites in or at the receptor binding site for immune evasion in human H3N2, and it is possible that this is what we see also in avian H3s.

A protein structure of the H3 hemagglutinin, where differences between the outbreak and the vaccine strains are mapped. For more information have a look at the paper in Molecular Ecology.

So, what can we learn from this? As always in science, each new study answers some questions but raises many more. First of all, what is the rate of antigenic drift in avian viruses, how do that differ among subtypes, and what does that mean in a functional and evolutionary context? How does this relate to long-term subtype dynamics and the role of herd immunity and heterosubtypic immunity in wild avian hosts? Second, it illustrates our lack of knowledge on the actual mechanisms of immunity –  despite low-pathogenic avian influenza viruses being gastrointestinal infections in waterfowl, we tend to study serum antibodies rather than mucosal antibodies or innate immune responses. Third, we have work to do as regards vaccination as a model for disease – are immune processes the same, and is protection similar?

Stay tuned – we will get back to this subject later.

If you want to read the study, it is available as Open Access:

Wille, M., Latorre-Margalef, N., Tolf, C., Stallknecht, D.E. & Waldenström, J. 2017. No evidence for homosubtypic immunity of influenza H3 in Mallards following vaccination in a natural experimental system. Molecular Ecology. [doi:10.1111/mec.13967]

http://onlinelibrary.wiley.com/doi/10.1111/mec.13967/full

 

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Life of (a) PI

From the movie Life of Pi – which is quite different from the Life of a PI.

This may come as a surprise for the PhD students and postdocs in the audience, but professors work too. We just do it differently than you. I know, it may seem as we are busy doing nothing, but in reality, we constantly juggle many tasks, some small, some large – some important, some superfluous. And many – to be honest – quite boring. Importantly, we do a lot of stuff so you don’t have to do them. Even if it doesn’t always seem so, we strive to make your life easier.

My main task is to make sure the research and the research group is functioning. This means setting overarching research goals, bringing in money, equipment, provide national and international contacts and all other things that allows students and staff to go about their business. It also entails a lot of hiring decisions, mentoring and scientific guidance – but equally much at the personal level, keeping folks happy and to be a partner in discussions of science and life.

But it doesn’t end there. Most professors teach (usually around 40-50% of their time), and even if lecturing is part of it, most time is plowed into planning courses, oversee curriculums, answering shitload of emails, meetings with colleagues, students, and the high and mighty folks at department and faculty levels. On top of that, there are the administrative duties, participating in various boards, special committees, answering questions, make budgets, report to agencies, sign invoices, hunt down people, etc.

But what about the science? Don’t professors do science? Well, we do, but not to the extent you may think. I rarely am the first author these days, rather I am the last and corresponding author. This means involvement in planning of the study, scientific guidance during the experiment, advice on stats and writing, steering the communication with coauthors, polishing of language, deciding which journal to submit to, and other things that needs to be done. It is also my responsibility to foster the larger context, the cooperation with other groups and apply for grants – and to train my students in the arts of becoming independent scientists.

Science and education thus make up the two main pillars of a professor’s duties. But there is a third, too, namely communicating science to the public. This is the aspect that varies most between professors: some do tons, some do little, and some do none. I kind of like doing it, which includes hosting this blog, but also to give lectures, or write popular pieces for ornithological magazines. It also includes expert advice to different government bodies, and answering media questions.

This mix of things sometimes make you feel you don’t do anything, or at least that you don’t do enough. So, as a part of inner reflection, I took a look at what I did yesterday. A Monday, just an ordinary Monday – one of many in the life of a PI.

A good way is to look at the emails, of which I received 70:

  • 1 was a confirmation of a submitted article (Yay!)
  • 10 dealt with the organization of an upcoming conference we are hosting (Check it out here)
  • 9 were from proMED (an invaluable resource for keeping abreast with disease information)
  • 1 dealt with a course that just started
  • 20 were either spam from fraudulent publishers, press-releases or commercials (delete, delete, delete)
  • 13 were from our traveling agencies regarding tickets for me and two of my incoming postdocs (no PA for professors)
  • 2 were from the HR department, dealing with hiring issues
  • 1 was from a research proposal from a collaborator in Bangladesh
  • 2 were from EFSA asking me whether I could participate in a panel on IAV (Parma is nice!)
  • 3 were from postdocs or postdoc candidates
  • 3 were from colleagues at the department on various small things
  • 5 were from admin staff to organize events to raise students (important, but not so yay)

Apart from dealing with these emails (I sent 22 emails, myself), I gave a short lecture in a course, wrote an advert for a position as lab technician, read two papers, looked over a poster for a conference, had a meeting with the Head of Department, spoke with three colleagues on different work-related issues (including hiring a PhD student), investigated why a grant hadn’t been paid, discussed the progress of postdoc project, tried to contact a funding agency (but didn’t succeed). All in all, quite a few things, but not much actual scienceing this day – more logistical/management stuff. This was just one day, and every day is unique. Most days I do more writing, be it grant writing or actual science – and other days I do more teaching. But at least you get the gist of it: a professor does many things. Even if frustrating at times, it still is the best job I can imagine.

Upcoming workshop – Ecology meets Epidemiology

dsc_04142On 22-23 March we will host a lunch-to-lunch workshop on disease ecology in Kalmar, Sweden. This workshop is arranged by  One Health Sweden, a network that promotes collaboration between researchers with interest in zoonotic infections and antibiotic resistance in Sweden and beyond.

At the Ecology meets Epidemioloy workshop we wish to gather scientists from biological, veterinarian and medical faculties to discuss the latest topics in disease ecology, ranging from animal ecology, disease epidemiology and immunology. We especially encourage young researchers (PhD students and postdocs) to come and present their work, meet senior researchers and exchange ideas with peers.

You can register now at the workshop’s homepage. From the abstracts we will select presenters for oral communications. Among the keynote speakers we have:

  • Nicolas Gaidet, Animal and Integrated Risk Management unit, CIRAD, France – An ecological approach to health: the case of avian-borne viruses
  • Elinor Jax, Max Planck Institute of Ornithology, Germany – Gene expression profiling of whole blood as a means of detecting an immune response in an avian non-model species
  • Gunilla Hallgren, National Veterinary Institute, Sweden – Anthrax in Sweden, from past to present
  • Lars Råberg, Functional Zoology, Department of Biology, Lund University, Sweden

More information and call for abstract can be found on the homepage – which will be updated as we go along.

It would be great to see you in Kalmar!

This duck and not that duck – what determines the susceptibility to infections?

Seasonal flu is just around the corner here in Sweden, with cases starting to rise week by week. And although the infection dynamic is fairly predictable at the population level, driven by both environmental factors and behavioral changes in the human population, it is not always easy to predict who will be infected or not, and whether an infection will result in mild or severe disease. Ultimately, this will depend on the exposure risk, the underlying condition of the individual, variation in specific genes in both the virus and the host and whether he/she has experienced previous infections, and in such case, how similar the current virus is to previous viruses. In short: complex interactions between the microorganism, the host, and the environment. These are things disease ecologists are interested in!

Unfortunately, for wildlife we know essentially nothing for most pathogens and hosts in terms of disease dynamics. And we know particularly little regarding the immune system and how variation in immune genes translates into protection against pathogens. If you read the literature, most studies look at the adaptive branch of the immune system, especially antibody mediated immunity and the diversity in the genes that make up the MHC loci. The MHC – or Major Histocompatability Complex – are among the most variable genes we know of, responsible for binding and presenting antigens to B- and T-cells and triggering immune processes. Although extremely interesting, they are but a part of the vast array of cells, proteins, and genes involved in immune processes.

In a recent paper, we dived straight into another set of genes: the beta-defensins. This is the first of several papers we will prepare on the subject. β-defensins are cool little proteins, actually kind of bad ass. Their main function is to interact with bacterial cell walls, where they create little pores and thereby interfere with cell homeostasis (think of Swiss cheese). β-defensins are part of the innate immune system and are ancient, present throughout the animal kingdom – and although some have evolved into new functions, such as toxins, the vast majority are involved in the fight against pathogens. Apart from directly killing invading bugs, they are involved in immune signaling and can also target some viral infections.

Our main study species is the Mallard, an important game bird species and the ancestor to domestic ducks – and the main reservoir host for influenza A virus in the Northern Hemisphere. Having studied disease dynamics in this host for a long time, we are now very curious about how variation in immune genes among Mallards translates into susceptibility to different diseases. Fortunately, the time is right for pursuing such questions, as the genome of the Mallard is available, making it easier to develop molecular tools to target specific genes. In a studied published a few weeks ago, we amplified and sequenced five β-defensin genes in a large number of individuals. First we studied these genes in a local population from Sweden, then we expanded it to cover specimens from all over the species’ range – from Europe, North America and Asia – and finally we sequenced the same genes in other species across the waterfowl phylogeny. This allowed as to ask how evolution has shaped β-defensin genes over different time scales. The results are summarized in the abstract, below:

All five genes showed remarkably low diversity at the individual-, population-, and species-level. Furthermore, there was widespread sharing of identical alleles across species divides. Thus, specific β-defensin alleles were maintained not only spatially but also over long temporal scales, with many amino acid residues being fixed across all species investigated. Purifying selection to maintain individual, highly efficacious alleles was the primary evolutionary driver of these genes in waterfowl. However, we also found evidence for balancing selection acting on the most recently duplicated β-defensin gene (AvBD3b). For this gene, we found that amino acid replacements were more likely to be radical changes, suggesting that duplication of β-defensin genes allows exploration of wider functional space. Structural conservation to maintain function appears to be crucial for avian β-defensin effector molecules, resulting in low tolerance for new allelic variants. This contrasts with other types of innate immune genes, such as receptor and signalling molecules, where balancing selection to maintain allelic diversity has been shown to be a strong evolutionary force.

Distribution of AvBD10 alleles across species, whereby coloured shading denotes mallard alleles and black shading denotes alleles found only in non-mallard waterfowl. Species with shading in the same vertical column share the allele denoted by that column. For more info see the original paper here.

To break this down into a more layman text, it means that most alleles are very old, dating back from before the different species split – evidenced by the same allele present in different waterfowl species separated by millions of years’ evolution. It also means that there is strong selection for maintaining function, that is, that mutations that change the amino acid composition largely are purged from the population. However, for one of the genes we saw evidence of balancing selection, where gene diversity in the population is favoured.

We hope this paper will be of interest for the evolutionary biology crowd, but we also see it as a first stepping stone into investigating how genetic variation translates into function. We are continuing with several lines of research, both in the lab and in experimental infections to measure how effective different allelic variants are against different pathogens, and where and how these genes are upregulated upon infection. This is very exciting research, but also a bit of a leap from what we have done in our lab before. It is a certain thrill to charter the unknown, and there’s tons of stuff to learn in order to do it well.

This paper was a collaborative effort involving four labs (Linnaeus University, University of Konstanze, University of Lund, and Wildfowl & Wetlands Trust):

Chapman, J.R., Hellgren, O., Helin, A.S., Kraus, R.H.S., Cromie, R.L. & Waldenström, J. 2016. The Evolution of Innate Immune Genes: Purifying and Balancing Selection on β-Defensins in Waterfowl. Molecular Biology and Evolution 33(12): 3075-3087.

 

 

On the air – participating in a podcast

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Yes.

I generally say yes to things. In fact, I like to say yes to things.

And although it is fun to say yes, it also comes with a cost, as the more you say yes, the more often you will be asked again. It is a dilemma, for sure, when time is a limited currency.

But saying yes is also a great way to do new stuff. Learn new things, meet new people. This autumn, for instance, I have participated in four different PhD defenses, in three different countries. Although a lot of work, it was great fun!

I also tend to say yes to scicomm stuff, ranging from giving talks to schools to participate in radio and newspaper articles. A few weeks ago I did something new: I said yes to participate in the Linnaeus University podcast ‘Snillen stimulerar’.

This pod is one of the university’s ways of disseminating academic knowledge to the public, but in a casual and entertaining way. If you click here, you can listen to a discussion on my favorite pathogens and diseases. Deadly stuff. A disclaimer, though: it is in Swedish.

So how was it? I enjoyed the experience, and I think the end product managed to be both informative and (surprisingly) fun. In this case, the two hosts – Ingeborg and Anders – took turns asking me questions, and making comments. They were brilliant, and had done their homework on my research, which made it all a rather smooth affair. The fun part is that you have to think on your feet and use a language less ladden with field specific scientific prose.

I hope you like it!

Come work with us – two positions as post doctoral fellows in disease ecology are available

dsc_04142

Funding comes and goes in mysterious ways, but right now I am in the fortunate position to advertise not one, but two postdoc fellowships in disease ecology at the Zoonotic Ecology and Epidemiology lab at Linnaeus University in Sweden. This is a great time to come join us! The lab currently consists of two senior researchers, four postdocs, two PhD students and one technician. We are also part of the centre of excellence Ecology and Evolution in Microbial Model Systems, a body of 50 or so researchers (PIs, postdocs, PhDs) that collaborate at the crossroads of disciplines. You will have a great time!

The project

The influenza A virus is a multihost pathogen with zoonotic potential, but most subtypes and genotypes are associated with wild birds, in particular dabbling ducks of the genus Anas. Since 2002, the Zoonotic Ecology and Epidemiology research group has worked broadly on host-pathogen interactions of this virus in a population of migratory Mallards in SE Sweden. Through systematic sampling for viruses we have built up a large collection of influenza A virus, and combined data on host and virus to build infection histories of the individual ducks. These data, together with targeted studies on movement ecology of Mallards, have enabled us to address disease ecology questions in this system. Currently, we seek to strengthen our research team with one or two Post Doctoral student(s) interested in disease ecology of IAV in a broad sense. The interested student should have research interests and prior experiences that align with the currently ongoing research, but could be focused either on evolutionary aspects of the virus (phylogenetics and evolution, or functional aspects of virus), or the consequences of the host of infection (either in terms of ecological costs, epidemiological modelling, or disease in a movement ecology perspective).

Qualifications

Requirements for the position are a strong record of disease ecology research with a PhD in either virology, veterinary medicine, ecology, molecular biology or similar. Other assessment grounds that would place the candidate at an advantage include a presentation of documented evidence of disease ecology research, preferably on influenza A virus, a high proficiency in written and spoken English, the ability to solve problems and to work independently, as well as interact in a research group.

Terms of employment

The position will be full time for 2 years. The proposed starting date is from the 1st of January 2017, but can be negotiated.

Apply?

Find out more and file your application on the following link.

Want to know more?

Check out our blog, read our papers, or send me a direct email (and to prove you are not a robot: jonas dot waldenstrom at lnu dot se)

Happy duck trap day!

1024px-birthday_candles

Yesterday, while fiddling around with some data for a government report I realized it was exactly fourteen years ago we opened the doors to the duck trap at Ottenby and started our surveillence program of influenza A virus in waterfowl. Fourteen years – and our fifteenth autumn season! That’s pretty good for a field series in ecology, and very, very good for influenza A virus surveillance in wild birds.

So happy duck trap day to all of you duck trappers (many, many), students (four PhD students, a good number of master- and undergraduate students), postdocs (four postdocs) and collaborators (from Sweden, The Netherlands, Germany, France, UK, US, Chile and elsewhere) – without you this wouldn’t been possible. And a big thanks to various funding agencies for supporting this work along the road.

Time flies, and so does the birds – and the viruses.