Adaptive Immunity

Like other invaders, protozoa stimulate both antibody- and cell-mediated immune responses. In general, antibodies control parasites in blood and tissue fluids, whereas cell-mediated responses are directed largely against intracellular parasites.

Serum antibodies directed against protozoan surface antigens may opsonize, agglutinate, or immobilize them. Antibodies together with complement and cytotoxic cells may kill them, and some antibodies (called ablastins) may inhibit their division. In genital infections of humans due to Trichomonas vaginalis, a local IgE response is stimulated. This triggers an allergic reaction that increases vascular permeability, permitting IgG antibodies to reach the site of infection and immobilize and eliminate the organisms.

In babesiosis the infective stages of the organisms (sporozoites) invade red blood cells. Infected red cells incorporate Babesia antigens into their membranes. These in turn induce antibodies that opsonize the red cells and cause their removal by phagocytosis. Infected red cells may also be destroyed by antibody-dependent cell-mediated responses. Macrophages and cytotoxic lymphocytes can recognize the Babesia antigen-antibody complexes on the surface of infected erythrocytes. T cell cytotoxicity may be important early in infection when the number of infected erythrocytes is small.

Intracellular parasites use many different and unique strategies to invade cells and inhibit intracellular killing. Most gain entry to a cell by employing host-mediated processes such as phagocytosis or induced uptake. Apicomplexans such as Toxoplasma and Cryptosporidium, however, actively penetrate cells using a system of adhesion-based motility called “gliding.” Once inside, these parasites reside in specially modified vacuoles. Protective immunity against apicomplexan protozoa, such as Cryptosporidium, Eimeria, Neospora, Plasmodia, and Toxoplasma species, is generally mediated by Th1 responses. For example, T. gondii is an obligate intracellular parasite whose tachyzoites grow within cells, especially macrophages (Figure 27-2). The parasites produce perforin-like molecules that permeabilize the cell membrane and permit the tachyzoites to escape and invade other cells. They penetrate these cells by “gliding” through a molecular junction in the cell membrane and do not trigger proper phagosome formation or maturation. Toxoplasma tachyzoites are therefore not destroyed since their “parasitophorous vacuoles” do not mature and fuse with lysosomes. Toxoplasma can grow inside cells in an environment free of antibodies, oxidants, or lysosomal enzymes. Toxoplasma-infected dendritic cells are attacked and killed by perforins from natural killer (NK) cells. However, the released parasites can then invade the NK cells! These infected NK cells, in contrast, are not effectively targeted by other NK cells.

The production of IL-12 and IFN-γ is essential for the early control of Toxoplasma infection. The toxoplasma actin-binding protein profilin is the ligand for TLR11. TLR11 on dendritic cells signals through the MyD88 pathway stimulating IL-12 and IFN-γ production. T. gondii cyclophilin also stimulates IL-12 production from dendritic cells through CCR5. The IL-12 and IFN-γ in turn trigger a strong Th1 response. Some antibodies are produced that, together with complement, destroy extracellular organisms and prevent its spread between cells (Figure 27-3). This response, however, has little or no influence on the intracellular forms of the parasite. The intracellular organisms are only destroyed by the IL-12-dependent Th1 response. Activated Th1 cells secrete IFN-γ in response to Toxoplasma ribonucleoproteins. This IFN-γ activates macrophages, permitting lysosome-vacuole fusion and killing the intracellular organisms. In addition, cytotoxic T cells can destroy Toxoplasma tachyzoites and Toxoplasma-infected cells on contact. T. gondii tachyzoites, however, may convert into a cyst form containing bradyzoites. The cysts are weakly immunogenic and do not stimulate inflammation. It is possible that this cyst stage is not recognized as foreign. As a result, the cysts persist indefinitely within tissues.

Th1-mediated responses resulting in the activation of macrophages are important in many protozoan diseases in which the organisms are resistant to intracellular destruction. One of the most significant destructive pathways in M1 cells is the production of nitric oxide (NO). Nitrogen radicals formed by the interaction of NO with other oxidants are lethal for many intracellular protozoa. However, protozoa are also experts in surviving within macrophages; for example, Leishmania, Toxoplasma, and Trypanosoma cruzi, can migrate into safe intracellular vacuoles by blocking phagosome maturation. Leishmania and T. cruzi can suppress the production of oxidants or cytokine production, whereas T. gondii can promote macrophage apoptosis. T. gondii tachyzoites can inhibit proinflammatory cytokine production by preventing nuclear translocation of NF-κB.

In Theileria parva infection (East Coast fever) of cattle, sporozoites can invade α/β and γ/δ T cells, as well as B cells. The parasite then activates NF-κB by continuously phosphorylating its inhibitor proteins Iκ-Bα and Iκ-Bβ (Chapter 8). The NF-κB thus persists, maintains the cell in an activated state, and prevents its apoptosis. The activated cells produce both IL-2 and IL-2R. As a result, a loop is established, by which infected cells secrete IL-2, which in turn stimulates their growth. As Theileria schizonts develop within lymphocytes, the infected cells enlarge and proliferate. Since the parasite divides synchronously with its host cell, there is a rapid increase in parasitized cells resulting in overwhelming infection and death. Some animals, however, may recover from infection and become solidly immune. In these animals, CD8+ T cells can kill infected lymphocytes by recognizing parasite antigens in association with MHC class I molecules. In susceptible animals, the parasites interfere with MHC class I expression.

Infection of chickens or mammals with Eimeria oocysts generally leads to strong, species-specific immunity that can prevent reinfection. This immune response inhibits the growth of trophozoites, the earliest invasive stage, within intestinal epithelial cells. This growth inhibition is reversible since the arrested stages can be transferred to normal animals and complete their development uneventfully. Studies in mice suggest that resistance to primary infection is mediated by multiple cell-mediated mechanisms that involve CD4+ T cells and their cytokines IL-12 and IFN-γ, macrophages, and NK cells. In contrast, resistance to secondary challenge is mediated by CD8+ T cells. In chickens, IFN-γ, tumor necrosis factor-α (TNF-α), and transforming growth factor-β (TGF-β), as well as intraepithelial CD8+ α/β T cells, appear to be essential for anticoccidial immunity. It is interesting to note that Eimeria-susceptible chickens express much more IL-10 in their intestines than do resistant chickens, both constitutively and after infection. Given that IL-10 promotes a Th2 bias, it is likely that this is the cause of reduced resistance in susceptible birds.

For many years it was thought that a common feature of many protozoan infections was premunition, a term used to describe resistance that is established after the primary infection has become chronic and is only effective if the parasite persists in the host. It was believed, for example, that only cattle actually infected with Babesia were resistant to clinical disease. If all organisms were removed from an animal, resistance was believed to wane immediately. Studies have shown that this is not entirely true. Cattle cured of Babesia infection by chemotherapy are resistant to challenge with the homologous strain of that organism for several years. Nevertheless, the presence of infection does appear to be mandatory for protection against heterologous strains. Babesiosis is also of interest since splenectomy of infected animals will result in clinical disease. The spleen not only serves as a source of antibodies in this disease but also removes infected erythrocytes. Splenic macrophages and dendritic cells trigger a Th1 response involving both NK cells and γ/δ T cells. They also generate NO. The absence of NO in splenectomized animals permits the disease to reappear.

Evasion of the Immune Response

Despite their antigenicity, parasitic protozoa survive within their host by using multiple evasion mechanisms acquired over many millions of years of evolution. For example, T. gondii can avoid neutrophil attachment and phagocytosis. T. parva invades and destroys T cells. Other protozoa such as the trypanosomes may promote the development of suppressive regulatory cells or stimulate the B cell system to exhaustion. Plasmodium falciparum can suppress the ability of dendritic cells to process antigen.

Parasite-induced immunosuppression may promote parasite survival. For example, Babesia bovis is immunosuppressive for cattle. As a result, its host vector, the tick Boophilus microplus, is better able to survive on infected animals. Infected cattle have more ticks than noninfected animals, and the efficiency of transmission of B. bovis is enhanced. It must be pointed out, however, that parasite-induced immunosuppression may kill the host as a result of secondary infection, so it is not always beneficial to the parasite. Death in bovine trypanosomiasis is commonly due to bacterial pneumonia or sepsis following immunosuppression.

In addition to immunosuppression, protozoa have evolved two other effective evasive techniques. One involves becoming less antigenic, and the other involves the ability to alter surface antigens rapidly and repeatedly. An example of a nonantigenic organism is the bradyzoite stage of T. gondii, which, as mentioned previously, does not appear to stimulate a host response. Some protozoa may mask themselves with host antigens. Examples of these include Trypanosoma theileri in cattle and Trypanosoma lewisi in rats, both nonpathogenic trypanosomes that survive in the bloodstream because they are covered by host serum proteins and are not regarded as foreign. T. brucei, a pathogenic trypanosome of cattle, may also adsorb host serum proteins or soluble red cell antigens, reducing its antigenicity.

Although reduced antigenicity may be considered the ultimate evasive technique, many protozoa, especially the trypanosomes, successfully employ repeated antigenic variation. If cattle are infected with the pathogenic trypanosomes T. vivax, T. congolense, or T. brucei and their parasitemia measured at regular intervals, the numbers of circulating organisms are found to fluctuate greatly. Periods of high parasitemia alternate regularly with periods of low or undetectable parasitemia (Figure 27-4). Serum from infected animals contains antibodies against trypanosomes isolated before bleeding but not against those that develop subsequently. Each period of high parasitemia corresponds to the expansion of a population of trypanosomes with a new surface glycoprotein antigen. The elimination of this population by antibodies leads to a rapid fall in parasitemia. Among the survivors, however, some parasites express new surface glycoproteins and grow without hindrance. As a result, a fresh population arises to produce yet another period of high parasitemia (Figure 27-5). This cyclical fluctuation in parasite levels, with each peak reflecting the appearance of a new population with new surface glycoproteins, can continue for many months.

The major surface antigens of these trypanosomes are known as variant surface glycoproteins (VSGs). These are the antigens targeted by host antibodies. The VSGs produced early in trypanosome infections tend to develop in a predictable sequence. However, as the infection progresses, the production of VSGs becomes more random. The VSGs form a thick coat on the surface of the trypanosome. When antigenic change occurs, the VSGs in the old coat are shed and replaced by an antigenically different VSG. Analysis indicates that these trypanosomes possess about 200 VSG genes, with an additional 1600 silent genes, of which two thirds are pseudogenes. Antigenic variation occurs as a result of repeated DNA breaking and repair, replacing an active VSG gene with one from the silent gene pool. Since only a small part of the tightly packed VSG is exposed to host antibodies, it is not even necessary for the complete molecule to change. Replacement of exposed epitopes by gene conversion is sufficient for effective variation (Chapter 17). Early in infections, complete VSG gene replacement occurs. Later on, partial replacement and point mutations can create new antigenic specificities. In some cases, the expressed VSG gene can be constructed as a mosaic from several archival pseudogenes. The potential for recombination-based variation is therefore absolutely enormous.

Trypanosomiasis is not the only protozoan infection in which variation of surface antigens occurs. It has also been recorded in infections by B. bovis, the plasmodia, and the intestinal parasite Giardia lamblia.

Since parasitic protozoa must evade the immune responses, it is not surprising that they preferentially invade immunosuppressed individuals. Organisms that are normally controlled by the immune response, such as T. gondii or Cryptosporidium bovis, can grow and produce severe disease in immunosuppressed animals. For this reason, acute toxoplasmosis and cryptosporidiosis commonly occur in humans immunosuppressed for transplantation purposes or for cancer therapy and in those infected with human immunodeficiency virus (HIV).

Adverse Consequences

The immune responses against protozoa may cause hypersensitivity reactions that contribute to disease. Type I hypersensitivity is a feature of trichomoniasis and results in local irritation and inflammation in the genital tract. Type II cytotoxic reactions are of significance in babesiosis and trypanosomiasis, in which they contribute to the anemia. In babesiosis, red cells express parasite antigens on their surfaces and are thus recognized as foreign and eliminated by hemolysis and phagocytosis. In trypanosomiasis, either fragments of disrupted organisms or possibly preformed immune complexes bind to red cells and provoke their elimination, causing anemia. Immune complex formation on circulating red cells is not the only problem of this type in trypanosomiasis. In some cases, excessive immune complex formation can lead to vasculitis and glomerulonephritis (type III hypersensitivity; see Chapter 30). Immune complex lesions are a marked feature of visceral leishmaniasis, as described previously.

Trypanosome infections may trigger an enormous increase in IgM-secreting cells so that very high levels of IgM are found in the blood of infected animals. Some of these antibodies are directed against autoantigens. These include rheumatoid factor–like molecules, and antibodies against thymocytes, single-stranded DNA, red cells, and platelets. In T. congolense–infected cattle, these polyclonally stimulated B cells are BoCD5+. As pointed out earlier, CD5+ B1 cells are of a different lineage to conventional B2 cells (Chapter 15). The mechanism of this polyclonal B cell activation is unknown.

It is probable that a type IV hypersensitivity reaction contributes to the inflammation that occurs when Toxoplasma cysts break down and release fresh tachyzoites. Extracts of T. gondii (toxoplasmin), if administered intradermally to infected animals, will cause a delayed hypersensitivity response (Figure 27-6).