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Will medical intervention such as vaccination prompt the evolution of more or virulent pathogens?

The evolution of drug resistant and vaccine escape mutants degrades the efficacy of chemotherapy and vaccination programmes. But treatment evasion is not the only detrimental evolution that can be prompted by medical and veterinary intervention. Virulence and transmission traits are the targets of animal and public health programmes, and they are intimately tied up with pathogen fitness.

We have recently argued that interventions that protect the host from death will relax selection against virulence. This can prompt the evolution of pathogens that, in the medium term, put unprotected hosts at greater risk and can in some cases make the population as a whole worse off. The framework underpinning this assertion is potentially relevant to a wide range of microparasites, but the specific fitness functions are grounded in our experiments on virulence of Plasmodium chabaudi in laboratory mice.

We are testing model predictions and experimentally investigating other relevant factors. In particular, we are testing whether semi-immunity can prompt the evolution of virulence, determining how coinfection impacts on transmission and virulence, and investigating possible bounds on virulence other than host death. We are also examining virulence in the mosquito vector, both because it is of interest in its own right, but also because it may impact on the evolution of virulence in the vertebrate.

As well as testing our theoretical framework, we hope our studies will provide data relevant to a large range of virulence evolution models, and generate data on a number of basic biological and ecological issues relevant to malaria natural history and control.

Group members involved: Andy Bell and Vicki Barclay.

Collaborators: Andrea Graham, Gráinne Long, Hamza Babiker, Richard Carter, Sylvain Gandon, Margaret Mackinnon, Sean Nee, Joanne Thompson

The following is a lightly edited version of the Background and Rationale section of the Wellcome Trust Programme grant, which was written in 2002.

Drug resistant and vaccine-escape (epitope) mutants erode the effectiveness of chemotherapy and vaccination. But their spread is not the only evolution that will be prompted by large scale medical and veterinary intervention. Virulence- and transmission-related traits are intimately linked to pathogen fitness, and are almost always genetically variable in pathogen populations. They can therefore evolve. They are also the target of control strategies. Understanding when medically-driven virulence and transmission evolution could be harmful or beneficial is the aim of this programme. Using an animal model of malaria, we will experimentally investigate determinants of pathogen life history evolution and experimentally evolve pathogens in the face of artifically enhanced immunity. Realisation of the potential of evolutionary biology to contribute to biomedical science(Williams and Nesse 1991; Ewald 1994; Futuyma 1995; Stearns 1999; Trevathan, Smith et al. 1999; Dieckmann, Metz et al. in press) requires that theoretical models are firmly grounded in the empirical reality of specific biological systems.

An illustration of the way in which public health interventions could drive clinically detrimental pathogen evolution is as follows. Classical models of the evolution of parasite virulence assume that excessively virulent mutants are eliminated by natural selection because they kill their host and therefore themselves. Excessively avirulent mutants also have low fitness because they are more rapidly cleared from their hosts or they fail to maximise their output of transmission propagules. Natural selection should optimise the balance between the costs of virulence (host death) and the benefits (immune evasion and host resource extraction)(May and Anderson 1983; Bull 1994; Ewald 1994; Lenski and May 1994; Read 1994; Frank 1996; Ebert 1999; Read, Aaby et al. 1999; Chao, Hanley et al. 2000; Regoes, Nowak et al. 2000; Lipsitch 2001; Dieckmann, Metz et al. in press). We have recently shown that the widespread use of vaccines that generate semi-immunity, rather than sterilising immunity, can lead to the evolution of increased virulence. This is because these vaccines reduce the selection against virulent parasite genotypes by reducing risk of host death (Gandon, Mackinnon et al. 2001). If this argument is correct, vaccine-driven virulence evolution will lead to increased mortality rates in unvaccinated individuals. The mode of action of the vaccine is key here. Anti-disease vaccines (those reducing in-host replication or parasite toxicity) will always generate this harmful evolution. In contrast, infection- or transmission-blocking vaccines should be selectively neutral because, when effective, they make the host an evolutionary dead-end. Any other medical intervention that reduces parasite replication or toxicity without eliminating the pathogens, such as subcurative chemotherapy or, in the veterinary context, enhanced genetic resistance through selective breeding, will also select for more virulent pathogens.

Our argument is mathematical, but there is evidence that vaccine-driven increases in virulence have occurred. Marek's disease virus (MDV), a herpes virus of chickens, has been getting increasingly virulent since WWII, apparently in response to vaccination (Witter 1997; Witter 1998; Biggs 2001; Witter 2001). In some cases, vaccines last about a decade before a more potent vaccine is required to deal with emergent, more pathogenic strains (Kreager 1998). These new strains are antigenically indistinguishable from their ancestors. The biology of MDV accords with our model, as does the anti-disease action of the vaccine. There is also good evidence that resistance evolution in rabbit populations in response to myxomatosis has led to increased myxoma virulence (Fenner and Fantini 1999). Thus, the evolutionary processes we discuss have apparently already had important consequences for animal health.

Could vaccine-driven virulence evolution occur in human diseases? Some diseases have been eradicated by vaccination before virulence evolution occurred (smallpox) or have natural histories different from that assumed in our model polio(Levin and Bull 1994), diptheria(Ewald 1996; Galazka 2000)). Any virulence increases that may have already occurred in other diseases may be hard to detect against a background of substantially improved general hygiene and medical treatment in wealthy countries. But, importantly, most vaccines in widespread use have been highly effective at preventing individuals transmitting disease: they reproduce the high levels of immunity achieved naturally. Future generations of vaccines will instead be directed against diseases for which natural immunity wanes rapidly or is rather poor even after repeated exposure. This is particularly true for malaria (Bruce-Chwatt 1963; Day and Marsh 1991; Marsh 1992; Bojang, Milligan et al. 2001; Richie and Saul 2002). We demonstrated, using evolutionary-epidemiological models of falciparum malaria, that widespread use of blood-stage and anti-toxin malaria vaccines could drive virulence upwards (Gandon, Mackinnon et al. 2001). This would put unvaccinated individuals at greater risk and erode the overall population-level benefits of vaccination. Once this has happened, vaccination could not be halted, because that would expose even more unvaccinated individuals to the virulent parasites.

This scenario is worrying, and its needs investigating. In particular, we need to know more about the likely timescales of such evolution. Our analyses suggest it would take longer than a clinical trial, and would probably occur over a few decades, as does the evolution of drug resistance. We also need to investigate ways to avoid it. Much of this work is theoretical, and ongoing with our collaborators. But equally pressing is the need to test the framework empirically (before we do the experiment on people). Our malaria models rely on evolutionary fitness functions revealed by our experimental work with Plasmodium chabaudi in laboratory mice(Buckling, Taylor et al. 1997; Taylor, Walliker et al. 1997; Taylor, Walliker et al. 1997; Taylor, Mackinnon et al. 1998; Taylor and Read 1998; Buckling, Crooks et al. 1999; Mackinnon and Read 1999; Mackinnon and Read 1999; Buckling and Read 2001; Timms, Colegrave et al. 2001; Mackinnon, Gaffney et al. in press; Read, Mackinnon et al. in press), and the bulk of this application is for further experimental work.

The theoretical framework, or our conclusion that vaccines could do medium-term harm to some sections of a population, may fail to withstand this experimental scrutiny. Falsifying either the framework or the conclusion would be an important advance. There are competing theoretical views (Bull 1994; Ewald 1994; Bergstrom, McElhany et al. 1999; Ebert 1999), some of which posit that the evolution of avirulence can be promoted by the right sort of vaccine(Ewald 1996). Our data will help evaluate these models too. Experimental tests of virulence theory are urgently required: the virulence literature has one of the worst ratios of theory to empiricism of any area of evolutionary biology. Moreover, much of the biology we will elucidate will be of interest in its own right. And, with the usual caveats about animal models, our experiments will also yield data relevant to malaria interventions in the field - not only medium term evolution, but also immediate consequences.

Malaria disease severity is determined by a complex interaction of host and parasite genetics, as well as factors such as previous exposure and socio-economics (Mbogo, Kabiru et al. 1999; Mackinnon, Gunawardena et al. 2000; Phillips 2001; Greenwood and Mutabingwa 2002; Miller, Baruch et al. 2002). Just as it is possible to study successfully the evolution of other traits with multiple causes, such as body size and host resistance, virulence evolution can be studied from a pathogen perspective if pathogen genes at least in part determine disease severity. For human malaria, direct evidence that parasites vary in virulence is actually rather limited. This is probably because high rates of recombination mean that virulent and avirulent strains are not easily recognised in the field. Some candidate virulence determinants in human Plasmodium species have been proposed(Marsh and Snow 1997; Hayward, Tiwari et al. 1999; Preiser, Jarra et al. 1999; Miller, Baruch et al. 2002), and overrepresentation of some parasite alleles among severe malaria cases has been reported(Engelbrecht, Felger et al. 1995; Robert, Ntoumi et al. 1996; Kun, Schmidt-Ott et al. 1998; Ariey, Hommel et al. 2001). However, experimentally demonstrating the involvement of particular genes has proved elusive, not least because virulence cannot be assayed in vitro. Nevertheless, a variety of indirect data point to genetic variation in Plasmodium virulence. First, deliberate infections of people demonstrated strain differences in virulence(James, Nicol et al. 1936; Covell and Nicol 1951). Second, various phenotypes believed to be encoded by parasite genes correlate with disease severity(Marsh 1992; Miller, Baruch et al. 2002), such as in vitro proliferation rates(Chotivanich, Udomsangpetch et al. 2000), rosetting(Carlson, Helmby et al. 1990; Rowe, Moulds et al. 1997), and cell selectivity(Simpson, Silamut et al. 1999). Finally, genetic variation for virulence has been readily uncovered in animal models of malaria(Yoeli, Hargreaves et al. 1975; Cox 1988; Mackinnon and Read 1999).

Taken together, these data suggest that, like other pathogens (Sibley and Boothroyd 1992; Lipsitch and Moxon 1997; Ebert 1998; Fenner and Fantini 1999; Pandey and Igarashi 2000; Ochman and Moran 2001; Read and Taylor 2001), Plasmodium has genetic variation in virulence. For some years now, we have been using P. chabaudi in mice to try to determine how natural selection will act on this variation. Genetically distinct clones differ in their virulence, measured as anaemia, weight loss and mortality(Mackinnon and Read 1999). These clone differences are consistent across a range of conditions such as dose(Timms, Colegrave et al. 2001), host sex (Mackinnon unpubl.), host immune status(Buckling and Read 2001), drug treatment(Buckling, Taylor et al. 1997), mosquito passage (Mackinnon unpubl.), the presence of competing genotypes(Taylor, Mackinnon et al. 1998; Timms 2001), and host genotype(Mackinnon, Gaffney et al. in press). Partial immunity reduces both virulence and transmission(Buckling and Read 2001). Crucially, there is strong evidence of fitness benefits to virulence: in the absence of host death, clones inducing more severe disease transmit better to mosquitoes(Mackinnon and Read 1999). Even in the absence of mosquito transmission, continual passage through mice leads to increasing virulence(Mackinnon and Read 1999). Thus, there is genetic variation in virulence and, in the absence of host death, more virulent clones have a fitness advantage and so would be favoured by natural selection. These experimental findings provide the empirical basis for our assertion that virulence evolution will increase if vaccines reduce host death rates(Gandon, Mackinnon et al. 2001).

In the P. chabaudi system, virulence correlates with rates of parasite proliferation(Mackinnon and Read 1999; Mackinnon and Read 1999), but it is unclear whether this is causal and if so, what the underlying mechanism is. Our models make no assumptions about any particular mechanistic basis of virulence - our arguments are based on functions relating the virulence phenotype to transmission and risk of host death. A range of mechanisms could be responsible. Margaret Mackinnon, a former Leverhulme-funded post-doc in the group, was responsible for the initial characterisation of the virulence phenotypes and their fitness consequences, and the preliminary data underpinning the work on semi-immunity in this proposal. While I plan to follow through the population and evolutionary consequences of this, elucidating the mechanistic basis of the virulence differences is the subject of Mackinnon's Dorothy Hodgkin Research Fellowship. She continues to work in my lab with my people and there will be substantial synergy between her efforts on mechanism and mine on virulence evolution.

Relevance of the animal model P. chabaudi is an ideal experimental virulence model for any microparasite whose biology accords with classic models of virulence evolution (pathogen-encoded virulence determinants, virulence-transmission trade-offs). How useful are our P. chabaudi data for understanding virulence evolution in P. falciparum? It is too early to say. P. chabaudi is certainly the best of the malaria models for investigating the questions we are interested in. Recent analyses of malaria therapy data suggests that, like P. chabaudi, a major determinant of severe disease and death is the inability to control the consequences of first peak of asexual parasites (Molineaux, Diebner et al. 2001). The natural host of P. chabaudi is the thicket rat, in which infections produce levels of virulence and kinetics of infection and infectiousness similar to those seen in mice(Carter and Walliker 1975; Carter and Walliker 1977). Host factors become key if they affect the relative virulence of different parasite lines; so far we have no evidence of that in different mouse strains (Mackinnon, Gaffney et al. in press) although we need to test that further. The models we wish to test are evolutionary, not co-evolutionary, and whatever the host, adapting to it will be the key evolutionary driver. If detailed information on the parasite determinants of falciparum severity becomes available, we will be well placed to test how relevant the P. chabaudi model is for P. falciparum. As ever, we need to recognise the possible limitations of animal models. However, it is also clear that the only way to investigate the evolutionary safety of population-wide interventions in human diseases is by combining work on mathematical models with data from animal models.

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