Parasitic infections are often genetically diverse, with hosts concurrently infected by more than one genotype. Crowding, where pathogen populations within a host are suppressed by the presence of competitor strains, could affect the health and infectiousness of individual hosts as well as the evolution of medically relevant traits such as virulence and drug resistance (Read & Taylor 2001). For example, selection for increased virulence is expected when a slower growing parasite is outcompeted by a faster growing, more virulent parasite (Bremermann & Pickering 1983; van Baalan & Sabelis 1995; Frank 1996; Gandon et al. 2001; Alder & Losada 2002). Similarly, the relative fitness of drug-resistant strains, and hence their rate of spread in a population, could be substantially enhanced when co-infecting drug-sensitive competitors are removed by chemotherapy (e.g. Hastings 1997, 2003, 2006; Mackinnon & Hastings 1998; Hastings & D'Alessandro 2000; Mackinnon 2005). Analogous evolutionary processes could affect the rate of evolution of epitope variants in response to strain-specific vaccination (Lipsitch & Samore 2002; Read & Mackinnon 2008).

Infections with the human malaria parasite Plasmodium falciparum frequently consist of more than one genotype (Anderson et al. 2000; Awadalla et al. 2001; Jafari et al. 2004; Walliker et al. 2005), and a variety of epidemiological evidence is consistent with crowding (Daubersies et al. 1996; Mercereau-Puijalon 1996; Arnot 1998; Smith et al. 1999; Bruce et al. 2000; Hastings 2003; Talisuna et al. 2006). In the rodent malaria model Plasmodium chabaudi in laboratory mice, there is a strong relationship between parasite virulence and crowding such that more virulent strains have a competitive advantage (de Roode et al. 2003, 2005a,b; Bell et al. 2006).

A number of biological mechanisms may underlie competition between strains within hosts (Read & Taylor 2001). One of these is immune-mediated apparent competition (Holt 1977), where increasing densities of one pathogen population elicits a host response that suppresses the population of another. T cell-dependent immune-mediated competition has been demonstrated in P. chabaudi (Råberg et al. 2006): in nude mice, which cannot produce mature T cells, competition was less severe than in nude mice reconstituted with T cells. Because many malaria vaccines currently under trial are aimed at inducing T cell-dependent responses, that experiment raised the question of whether vaccination might exacerbate in-host competition and thus affect pathogen evolution, for instance by strengthening selection for competitive ability and hence virulence.

The effects of immunity on in-host competition are unlikely to be simple. The immune response to Plasmodium infection has both pathogen genotype-transcending (non-specific) and genotype-specific components. Protection is generally thought to become more specific during later stages of infection (Jarra & Brown 1989; Buckling & Read 2001; Mackinnon & Read 2003; Stevenson & Riley 2004; Martinelli et al. 2005; Cheesman et al. 2006). Thus, in contrast to non-specific immunity that could generate immune-mediated apparent competition, specific immunity could, in principle, act to alleviate competition (Råberg et al. 2006). Here, we extend the study by Råberg et al. (2006) by focusing on a specific subset of T cells, in order to further investigate the importance of immunity in determining competitive outcomes within hosts.

T cells can be divided into two major categories, CD4⁺ and CD8⁺ cells. It is well established from both experimental animal models and field studies in humans that the CD4⁺T cells play a pivotal role in the development of blood stage immunity to Plasmodium infection (Good & Doolan 1999; Pombo et al. 2002). They are initially required to produce cytokines that amplify the phagocytic and parasitocidal response of the innate immune response and later on to dampen this response to limit immunopathology. As the response becomes more adaptive they are required to help B cells produce antibodies that are essential for parasite clearance (Urban et al. 2005; Stephens & Langhorne 2006).

Since the CD4⁺T cells have been described as having such a crucial role in natural immunity to the blood stage of infection, and vaccine programmes strive to mimic and enhance this response (e.g. Stephens & Langhorne 2006), we have begun to investigate the specific role of these cells during competition in mixed infections of P. chabaudi. Specifically we looked at the acute phase of infection, where any interaction between the parasite and the host immune response could strongly influence host health (Urban et al. 2005). We chose two parasite genotypes that had been shown previously to differ in competitive ability and compared the extent of competition in immuno-competent and CD4⁺T cell-depleted mice. We hypothesized two possible scenarios: (i) if T cell-dependent immunity induces a non-specific response, then a numerically subdominant clone would experience a stronger immune response in a mixed infection than when on its own. Thus, competition should be eased in CD4⁺T cell-depleted mice. (ii) If the immune response is largely clone-specific and primarily elicited against the numerically dominant clone, then CD4⁺T cell depletion may exacerbate other forms of competition, such as competition for limited resources such as red blood cells.

Mice treated with the anti-CD4⁺T cell antibody were successfully depleted of CD4⁺T cells, both prior to parasite challenge and during the whole course of the experiment (figure 1). The CD4⁺T cell depletion resulted in more parasites of both clones (figure 2a,b; table 1).

We found no evidence that the CD4⁺T cells enhanced competition during mixed genotype infections with P. chabaudi (figure 2a; table 1). Specifically, during the peak stages of acute infection (days 6–8) suppression was independent of the CD4⁺T cells (figure 2c; table 3). After the peak of infection (day 9+), the CD4⁺T cells acted to alleviate competition such that upon their removal competitive suppression was enhanced (figure 2d,e; table 3). In addition, the presence of CD4⁺T cells did not cause suppression of the dominant genotype (figure 2b; table 4).

The immune response to Plasmodium infection has both pathogen genotype-transcending (non-specific) and genotype-specific components, with protection becoming more specific during later stages of infection (see §1). Here, we found that after the peak of acute infection (day 9 onwards), there was no competitive suppression of DK parasites in intact control mice; whereas in CD4⁺T cell-depleted mice, there was still evidence of competition (figure 2d,e; table 3). Both clones did better in depleted mice, probably owing to an impaired early antibody production through lack of T cell help and possibly the reduced recruitment and activation of macrophages for the uptake of infected cells. Thus, in normal hosts, a largely clone-specific adaptive immune response towards a numerically dominant genotype may act to alleviate competition by regulating clonal populations and limiting other forms of competition, e.g. competition for red blood cells.

During the peak of infection (days 6–8), competition was CD4⁺T cell independent so that the extent of competitive suppression of clone DK was similar in intact control and CD4⁺T cell-depleted mice (figure 2a,c; table 3). A number of biological mechanisms could be the proximate cause of competitive suppression during the peak of infection. First, there may be direct interference between two infecting strains. This has not yet been demonstrated in any parasites, but pathogenic bacteria can produce allelopathic substances that actively suppress competitors (Riley & Gordon 1999), and competing viruses can produce interference molecules (Hart & Cloyd 1990). Second, the competition may be influenced by non-specific components of the innate immune response (CD4⁺T cell independent). Third, there may be competition for resources as genotypes infecting mice simultaneously must divide the available red blood cells and other resources such as blood glucose between them (Hellriegel 1992; Hetzel & Anderson 1996; de Roode et al. 2005a; Gurarie et al. 2006). The CD4⁺T cell-depleted mice were more anaemic than control mice (figure 3; table 5), so that if red cells are limiting, there is more potential for competition for that resource in depleted mice. Mathematical models have suggested that during mixed infection, the proximate cause of competitive advantage may be attributable to an earlier and wider red blood cell preference of dominant genotypes (Hellriegel 1992; Gravenor et al. 1995; McKenzie & Bossert 1997; Jakeman et al. 1999; Mason & McKenzie 1999; McQueen et al. 2004; Antia et al. submitted). Because these predictions are based on the data from the rodent malaria model, they could be tested directly by transferring red blood cells of different ages into a single mouse and determine their loss following infection, or indirectly by measuring competition in untreated mice and mice treated with erythropoietin (Suzuki et al. 2006).

Our conclusion that competition is not CD4⁺T cell mediated apparently contradicts the recent finding of T cell-mediated apparent competition (Råberg et al. 2006). In that study, the authors looked at mixed infections with P. chabaudi in nude mice (which lack the ability to produce mature T cells) and compared the extent of competition with that in nude mice reconstituted with T cells. There was still pronounced competition in all animals, but there was some alleviation of competitive suppression in nude mice towards the end of the acute phase of infection, when the initial wave of parasitaemia was waning. This period corresponds roughly to days 9–14 in figure 2. A number of experimental differences could explain the contrasting results of Råberg et al. (2006) and the present study. First, different mouse strains were used in the two studies and host genotype has previously been shown to quantitatively affect the outcome of competition (de Roode et al. 2004). Second, different pairs of clones were used and P. chabaudi clone can induce different levels of strain-specific immunity (Cheesman et al. 2006). Third, there was a difference in the method used to modulate T cell-dependent immunity. Nude mice lack the ability to produce any mature T cells, including both CD4⁺ and CD8⁺T cells. The role of CD8⁺T cells during malaria infection in mice is still unclear (Lamb et al. 2006), but it could be that they are involved in the relatively small component of competition that was shown to be immune-mediated competition in reconstituted nude mice (Råberg et al. 2006). In addition, the repertoire of serum antibodies (including both natural antibodies and antigen elicited antibodies) in the CD4⁺T cell-depleted mice will be different from that in nude mice. Nude mice grow up producing only T cell-independent antibodies, while in the CD4⁺T cell-depleted mice there will be both T cell-independent and persisting T cell-dependent antibodies (produced by existing plasma cells in the bone marrow) and these may cross react with the parasite.

Taken together, the present study and that of Råberg et al. (2006) show that the effect of T cell-dependent immunity on competition is relatively weak, and may be either positive or negative depending on specific details of host and parasite. Rather than further dissection of any immune mechanism-mediating competition, one could use this malaria model system to look at the strength of competition in hosts immunized by a variety of different candidate vaccines towards the blood stage of infection. Meanwhile, the result we report here suggest that vaccines that enhance CD4⁺-dependent immunity will not increase the selection in favour of virulence arising from in-host competition.