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

 

Of chickens, wild birds and men – host specificity in Campylobacter jejuni

Rule number one in the kitchen: be wary of chickens! Improperly handled, this meat may spice up your dish with unwanted avian gut bacteria. The most notorious chicken bug is Campylobacter jejuni – which gives you really, really bad gastroenteritis (or ‘shits’ as most of you would say).

C. jejuni is a quite common bug. At the poultry flock level, prevalence vary between 0 and 90% depending on which time of the year it is (more in summer months), which country we are talking about (less at northern latitudes), and of course the hygiene level of the farm in question (greasy farms get more bugs). However, nice Campylobacter-free chickens may be soiled with bacterial cells from infected birds during the long winding road from the farm, through the various stations in the abattoir (de-feathering stations, rinsing etc.) and to the retail level and end-consumer.

In our research we have addressed wild birds as hosts for campylobacters. Over the years we have spent a lot of effort to find out which bird species that are carriers of campylobacters, and which that are not. And what kind of differences there are between bacteria from different bird species. Earlier this year, we published a study in Molecular Ecology, where we genotyped a large collection of C. jejuni collected from Sweden, England and Australia. For a full view, down-load it here: http://onlinelibrary.wiley.com/doi/10.1111/mec.12144/full

We used multilocus sequence typing (MLST), which is equal to sequencing parts of seven different housekeeping genes distributed around the bacterial chromosome. Each unique allele gets a number, and the combined row of numbers of the seven loci is used to create a sequence type (ST). A sort of fingerprinting, you could say. And a very handy technique for C. jejuni, as it is one of the most recombinatory bacteria we know of; tree-based methods for inferring relationships don’t work as good on campylobacters.

We found two things: First, C. jejuni populations in wild birds have very different genetic structure from C. jejuni in farm animals. In the figure above, you see how all human and food-animal C. jejuni populations cluster together, and where the different wild bird hosts have distinct populations of bacteria with long branch lengths. Second, we found strong patterns of host specificity.

Have a look at the picture again. If you look carefully, you will see that dunlins in Sweden and sharp-tailed sandpipers in Australia have more or less similar C. jejuni, despite huge geographic distances! Same goes for black-headed gulls and silver gulls, very similar to one another, but very different from the waders! And have a look at the blackbird – introduced to Australia by acclimatization societies in the 19th century they seem to have retained similar genotypes of C. jejuni that modern blackbirds have in Europe! Remarkable!

This really tells you of host adaptations – there are certainly differences in the enteric environment of different bird species, and in their diets, but there may also be differences in ecology that affects transmission properties, or survival in the environment. And why is all this important? Well, it says something about the peculiarity of current food animal C. jejuni. In these hosts, C. jejuni are more genetically similar and have a larger host range, suggesting that particular features involved in survival and transmission in the farm environment has caused expansion of particular genotypes.

In the future, identifying these properties are key. Hopefully we could do that with our wild bird campylobacters. But in the mean time, wash your hands and cook your chicken properly.

Jonas Waldenström

http://onlinelibrary.wiley.com/doi/10.1111/mec.12144/full