Host's immune response

Host's immune response

An integral part of the host's immune response is recognition of parasitic antigens that are shed and expressed on the cell surface. These antigens are the

immunodominantepitope in trypanosomiasis, and without host recognition the proliferating parasite will quickly overwhelm and kill the host. In this sense, the parasite actually depends on the host's immune system for its survival, which is an example of the importance of co-evolution between pathogens and hosts. While many aspects of the innate and adaptive immune response are important in controlling infection, the key aspects of the anti-trypanosome response are: a rapidly executed type 1 pro-inflammatory response that leads to antigen-specific antibody production, and macrophage activation that leads to parasite killing by TNF, NO and IFN. The way that trypanosomes evade these immune responses is based on the densely packed Variant Surface Glycoproteins (VSG) that coat theparasite.

All pathogens that depend on humans for survival are subject to our immunological response, which attempts to kill and eradicate them. These pathogens must be able to evade, and even suppress, with the human immune system in order live. However, they must also proliferate and disseminate before they completely overwhelm the host and kill it. Survival of a successful African Trypanosome, a protozoan parasite, depends on its ability to switch its immunodominant epitope(s): the glycoprotein coat on the cell surface. Through a mechanism called antigenic variation, the African trypanosomes, as well as other protozoan parasites such as Giardiaintestinalisand Plasmodium species, can successfully establish chronic infections in humans.

Trypanosomacruziis mainly found in South and Central America and causes Chagas disease. Trypanosomabruceirhodesienseand bruceigambienseare found in Africa, and cause acute and chronic forms of Sleeping Sickness. These diseases are characterized by waves of fever that appear to mirror the fluctuations in levels of parasitemia, which are caused by the cycles of immune recognition of new surface epitopes on the parasite

Host-parasite interactions

An integral part of the host's immune response is recognition of parasitic antigens that are shed and expressed on the cell surface. These antigens are the immunodominant epitope of all species of trypanosomes. Trypanosomes in the human blood involve both intracellular and an extra cellular stages of the life cycle. Antigens on the surface of the parasite are released upon entry into cells, and these antigens can activate the complement system and can be phagocytosed by DC's and macrophages. During the intracellular stage antigens from the parasites appear on the cell surface, and are also shed when the parasite bursts the RBC, moving into the extracellular environment. The antigens stimulate the rapid activation of NK cells and macrophages, which produce IFN-gamma, activating monocytes to produce type 1 cytokines and cytotoxic compounds, such as NO. After uptake by

Figure 2 macrophages and DCs antigen is presented to naïve Tcells in the presence of IL-12 and IL-18, and this polarizes a type 1 immune response. Activation of B cells leads to antigen-specific antibody synthesis, and these antibodies, in conjunction with sustained monocyte activation, control parasite proliferation. However, later in infection it important for the host to be able to control the type 1 immune response in order to reduce injury to its own tissues. Usually, after a few days the host immune system is able to control the parasite through VSG-specific antibodies and through micobicidal molecules released by monocytes. But because of the frequent shift in the surface VSG epitopes, the parasite is able to continually outrun the host's immune response.

Antigenic variation

Variant Surface Glycoproteins In trypanosomes the immunodominant antigens are the VSG proteins that packed tightly together to coat the parasite (figure 3). The VSG protein occurs in membrane form (mfVSG) on the surface of infected cells and the extracellular parasite, and in soluble form (sVSG) as a result of shedding. VSG is synthesized, transported and anchored to the plasma membrane where it is covalently linked to glycoslphosphatidylinositol (GPI). During infection a PLC enzyme becomes activated and cleaves the GPI Figure 3

anchor, releasing soluble VSG that retains the glycosylinositol phosphate (GIP) and leaves dimyristoylglycerol (DMG) on the membrane. Studies have shown that both mfVSG and sVSG have similar macrophage activating properties. However, mfVSG more effectively activates macrophages that produce IL-1 and IL-12, which leads to subsequent activation of more macrophages and to a Th1 cell polarization (Coller 2001). VSG genome The genomic structure of Trypanosome species are diploid for housekeeping genes, but are aneuploid for small, unique chromosomes, which are 200-430kb in size and present in each cell. These small chromosomes, called minichromosomes, encode many genes for surface glycoproteins (Lex HT van der Ploeg 1990). Usually only one of these genes is transcriptionally active at a given time, which allows the parasite to completely switch its protein coat from one VSG variant to another in only a few days. However, it is well documented that VSG genes are under the control of promoters that are in subtelomeric regions of DNA, not in the centers of minichromosomes where the majority of VSG are found. This suggested that VSG genes are mobile and must somehow get to the expression sites in telomeres(Miguel Navarro 2007). Transcription and translation of genes that cause antigenic variation are a result of rearrangements at the gene expression site. This occurs in two main ways: the first involves duplicative transposition, allowing a silent VSG gene from the central section of the minichromosome(s) to be transposed into one of 20

Figure 4 expression sites in the telomere (figure 4, 2). The second mechanism, called telomere exchange, allows genes that have already been transposed into telomeres to be activated by deletion of old gene (Figure 4, 3). The placement of genes into transcriptionally active sites on other telomeres efficiently uses the activated promoters of the VSG genes.

These events allow the trypanosomes to switch their protein coats in a mutually exclusive fashion every few days. In addition to these recombination events, genetic diversity is added into the VSG genes though silencing different of telomeric expression sites, hypervariable region base pair variation, point mutations, and post- transcriptional alterations of the mRNA (Gloria Rudenko 1998). Expression sites In any given coding region of a telomere multiple expression sites are under the control of one promoter region. Early in infection, exchange of genes between expression sites is an efficient mechanism that allows trypanosomes to include genetic variation in the VSG proteins with a minimal amount of “effort” expended. Instead of recombining with a VSG gene from a minichromosome the DNA can simply make an in situ switch between expression sites already located in the telomere. Each region of telomeric expression sites (figure 6), and the active expression site is where transcription of the VSG RNA begins. At any given time, only one expression site can escape silencing and transcribe genes (fig. 5). However, because only 20 expression sites exist in the telomeres only 20 VSG genes can be accessed at one time by this mechanism. In situ switching is thought to be controlled by transcriptional elements that mediate silencing of certain expression sites. In situ switching mechanisms are not completely clear yet, but they are known to cause the activation of new expression sites and silencing of old ones. Expression site silencing seems to have a relationship to the expression site's position on the telomere, such that the farther away it is from the end of the chromosome the easier it overcomes silencing (Gloria Rudenko 1998; Inês Chaves 1999).

Additionally, in the VSG expression sites there are regions that can add

genetic diversity to the gene repertoire during gene conversions. These hypervariable (HV) regions change due to selective pressure from the host's immune system. These regions are the main epitopes that are recognized by B cell and T cell receptors, so hypermutation during gene recombination may allow them to evade detection by these cells. Another source of genetic diversity of the encoded VSG genes are point mutations that occur during gene replication, but the specific importance of these mutations in the genetic diversity of the VSG proteins is not yet known (Alan G babour 2000). One of the hallmarks of the trypanosome protein coat is that only one VSG variant is expressed at one time, except in the transient mixed coat that appears during the switch from one coat to the other. In fact, no trypanosome with a stable

mixed coat of proteins has been confirmed. This suggests that expression of VSG genes must be mutually exclusive. Two mechanisms are proposed for this interaction: first, two expression site promoters may be active at one time, but one of them may “sense” the over-expression of the other and shut off. Second, there may be a physical restriction on the co-expression of activation site promoters (Gloria Rudenko 1998).

Evasion of the immune response: perturbation and escape

The current view of immune response by trypanosomes is that B-cells, activated by both by T-helper-independent and T-helper dependent mechanisms, and monocytes are the primary effector molecules. The VSG is the immunodominant epitope in trypanosomes, but free trypanosome DNA and fragments of the VSG proteins that are shed during the immediate immune response are important in immune regulation. As is described above, these molecules are well known for their ability to evade the human immune system by antigenic variation. However, recently these molecules have been shown to be able to actively deregulate the immune response. For example, there is a time-dependent mechanism for controlling macrophage activation, wherein trypanosomes that quickly shed many molecules upon host invasion can perturb activation. Studies have demonstrated that high levels of sVSG exposure to macrophages before IFN-gamma priming results in a substantial decrease in production of TNF-alpha and NO, which are potent cytotoxic molecules and critical for controlling parasitemia. IFN-gamma production is further impaired and results in the inadequate priming of macrophages. This is associated with the alteration of the STAT-1 signaling pathway that begins at the IFN-gamma receptor on macrophages. These altered macrophages are limited in the cytokines that they produce and in their APC capabilities, thus disturbing other facets of the adaptive immune response (JM Mansfield 2005; Terry Pearson 2005).

In addition to activation of B cells by T helper 1 cells surface proteins of the trypanosome protein coat cross-link B cells and initiate T-independent activation events. Antigenic variation allows the parasite to evade humoral immunity by slowing both these methods of B cell activation. As new VSG genes are expressed, transcribed and trafficked to the surface of the parasite the stability of the old coat results in the co-expression of the new and nascent VSG proteins for about 48 hours during the process of antigenic variation. The TI B-cell response, primarily characterized by secreted IgM, to the mosaic trypanosoma is compromised relative to the response seen in hosts with homologous VSG proteins on the protein coat (Melissa E. Dubois 2005). Additionally, T helper cells specific to the new surface coat proteins must be activated and polarized to express T-helper1 surface markers. After the protein coat has been completely replaced by the new VSG proteins, another B-cell response can be initiated against the new protein coat, however the lag-time for the B cell population to create new VSG-specific antibodies prevents the host immune system from immediately controlling the infection and the parasites proliferate in the blood stream. However, after a few days the antibody response becomes fine-tuned to the new coat of VSG proteins on the parasite. But by this time, a new set of VSG proteins are being made and the process begins all over again.

T-helper cells appear to recognize VSG peptides from only a limited sub region of the molecule. These regions happen to be those that are hypervariable (HV) and not conserved in the genome. Thus, antibody and T cells are a selective pressure for the variation in the regions of the VSG molecule's genome. The parasite has co-evolved with the human immune system, and possesses the ability to add genetic diversity in these regions in order to try to avoid the adaptive immune response by B and T cells. Successful parasites are those that can chronically infect the host without killing it. Hence, the switch to a type 2 anti-inflammatory T cell response is important to restrain the tissue damage caused by NO, TNF-alpha and IFN-gamma. as infection progresses. Although it is in the interest of the host to balance out the type 1 response with a type 2 response, it is a precarious situation because the immune system must maintain cytotoxic killing of parasites without killing itself. The parasite as well must participate in this balancing-act in order to counter the cytotoxic effects of the effector cells. Hence, the parasites attempt to skew the balance toward the type 2 response by producing higher levels of type 2 cytokines. The switch of T cell subsets is induced by IL-10 and IL-4, secreted from monocytes with alternative activation phenotypes, may be mediated by sVSG and its effects on macrophage activation (Sternberg 2004).


As soon as a parasite enters its human host a type of race commences between the host's immune system and the parasite. The immune system tries to control the proliferation of the parasite while the parasite attempts to perturb the immune response in order to proliferate inside of the host. The winner of this race determines clinical outcome of parasitic disease, and if the parasites win the host will eventually die. In trypanosomiasis one characteristic of a controlled infection is a patterned cycle of fevers, each wave occurring as the parasite makes different immunogenic surface proteins. Both attached and free proteins from the Trypanosome VSG protein coat interrupt macrophage activation, B cell activation, and humoral immunity.

The competition for survival between the genomes of the human host and the trypanosome often favors the parasite, not just in the individual, but also globally. For example, in 2004 a form of trypanosome, T.evansi, was discovered to have jumped to human hosts in India, a region previously unaffected by trypanosomiasis. Characteristically, the parasites disappeared on day 2 of infection, but reappeared on the tenth day accompanying a fever in the patient (2005). This, and the continued burden of Chagas disease and Sleeping Sickness, makes finding a lifelong vaccine a pressing issue in public health. There are many groups hunting for this vaccine, but the main hurdle to overcome - identifying conserved trypanosome antigens that elicit an antibody response - is yet to be surmounted.

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