The relative contribution of antibodies, CD4+ and CD8+ T cells to sporozoite-induced protection against malaria.

Protective immunity against Plasmodium yoelii, induced by sporozoite immunization, was investigated using a quantitative method based on the measurement of plasmodial ribosomal RNA in the liver of sporozoite-challenged mice. The relative importance of the different immune mechanisms induced by sporozoite immunization was determined by evaluating quantitatively the anti-parasite activity of antibodies, CD4+ and CD8+ T cells. The role of antibodies was determined by passive transfer of immune sera to naive mice. The transfer to mice of sera obtained after a single immunizing dose reduced the liver stages by 47%. The respective contribution of CD4+ and CD8+ T-cell subsets was determined in B10 (H-2b) mice, treated with a monoclonal antibody (mAb) which inhibits B-cell maturation, and subsequently immunized once with irradiated sporozoites. These mice produced low levels of anti-sporozoite antibodies, but were capable of inhibiting the development of liver stages as efficiently as non-manipulated immunized mice. Administration of either anti-CD4 or anti-CD8 mAb to these mice, did not significantly decrease their capacity to inhibit the development of liver stages. We only observed a significant loss of immunity when the mice were depleted in vivo of both CD4+ and CD8+ T cells. In contrast to earlier studies, we found that the induction of protective immunity is not a phenomenon restricted to a few strains of mice having a particular genetic make-up. The apparent non-responsiveness observed in some strains of mice can be overcome by using larger immunizing doses.     

Priming of CD8+ T cell responses following immunization with heat-killed Plasmodium sporozoites

Protective immune responses against malaria are induced by immunization with radiation-attenuated Plasmodium sporozoites. In contrast, non-viable, heat-killed sporozoites do not induce protection, emphasizing the requirement for live parasites to achieve effective immune responses. Using an experimental system with CD8+ T cells from T cell receptor-transgenic mice, we analyzed the primary CD8+ T cell responses elicited by heat-killed inactivated sporozoites. We found that the numbers of specific CD8+ T cells induced were much lower compared to when immunizing with attenuated sporozoites; however, the kinetics of activation and the phenotype of these T cells were similar in both groups. Despite their low frequency after priming, high numbers of specific CD8+ T cells were observed after boosting with a recombinant vaccinia virus. Upon induction of the recall response, the same level of protection was observed when either heat-killed or attenuated sporozoites were used for priming. We propose that live parasites are not critical for the induction of memory T cell populations against the malaria liver stages.     

Class II-restricted protective immunity induced by malaria sporozoites

The irradiated-sporozoite vaccine elicits sterile immunity against Plasmodium parasites in experimental rodent hosts and human volunteers. Based on rodent malaria models, it has been proposed that CD8+ T cells are the key protective effector mechanism required in sporozoite-induced immunity. To investigate the role of class II-restricted immunity in protective immunity, we immunized beta2-microglobulin knockout (beta2M-/-) mice with irradiated Plasmodium yoelii or P. berghei sporozoites. Sterile immunity was obtained in the CD8+-T-cell-deficient mice immunized with either P. berghei or P. yoelii sporozoites. beta2M-/- mice with the BALB/c (H-2d) genetic background as well as those with the C57BL (H-2b) genetic background were protected. Effector mechanisms included CD4+ T cells, mediated in part through the production of gamma interferon, and neutralizing antibodies that targeted the extracellular sporozoites. We conclude that in the absence of class I-restricted CD8+ T cells, sporozoite-induced protective immunity can be effectively mediated by class II-restricted immune effector mechanisms. These results support efforts to develop subunit vaccines that effectively elicit high levels of antibody and CD4+ T cells to target Plasmodium pre-erythrocytic stages.     

Peptide-based subunit vaccines against pre-erythrocytic stages of malaria parasites.

Malaria currently ranks among the most prevalent infections in tropical and sub-tropical areas throughout the world with relatively high morbidity and mortality particularly in young children. The widespread occurrence and the increased incidence of malaria in many countries, caused by drug-resistant parasites (Plasmodium falciparum and P. vivax) and insecticide-resistant vectors (Anopheles mosquitoes), indicate the need to develop new methods of controlling this disease. Experimental vaccination with radiation-attenuated sporozoites can protect animals and humans against the disease, demonstrating the feasibility of developing an effective malaria vaccine. However, vaccines based on radiation-attenuated sporozoites are not feasible for large scale application due to lack of in vitro culture system. Therefore, the development of peptide-based subunit vaccines has been undertaken as an alternative approach. Synthetic peptides containing defined B- and T-cell epitopes of different antigens expressed in sporozoites and/or liver stages have been used as subunit vaccines in experimental animal models. They have been shown to be highly immunogenic and capable of inducing protective immunity mediated by antibodies, as well as CD4+ and CD8+ T-cells.     

Hepatic phase of malaria is the target of cellular mechanisms induced by the previous and the subsequent stages. A crucial role for liver nonparenchymal cells

Both the sporozoites and the erythrocytic stages can modulate the hepatic phase by cytokines, notably IFN-gamma, TNF and IL-6, either directly or as a result of a cascade of events, and by MHC-restricted and antibody-dependent cell-mediated cytotoxicity. The role played by CD8+ T cells in inducing protective immunity against pre-erythrocytic stages is clearly established. The potential interest of triggering peptide-primed CD4+ T cells has to be considered regarding protection. Indeed, CD4+ T cells induced by the non-repetitive part of the CS protein of Plasmodium yoelii are protective, by eliminating malaria from hepatocytes. The crucial role of the liver NPC has to be emphasized, their participation in TNF schizonticidal effect and in ADCC mechanisms being strongly supported by our data.     

Granule-dependent killing of Toxoplasma gondii by CD8+ T cells

Immunization of mice with live bradyzoites of a low-virulent Beverley strain of Toxoplasma gondii has been shown to increase CD8+ T-cell mediated immunity against a highly virulent RH strain. We found that preimmunization with an RH homogenate further enhanced this immunity. Using this model, we investigated the mechanism of CD8+ T-cell mediated protection against T. gondii infection. Splenic cells from mice immunized with RH homogenate and live bradyzoites stimulated apoptosis of RH-infected J774A.1 macrophages in vitro, and at the same time, the immunization significantly suppressed the proliferation of parasites within macrophages, as assessed by measuring 3H-uracil uptake by the parasites. Splenic cells from the immunized mice produced larger amounts of interferon-gamma (IFN-gamma) than did naive splenic cells; however, the production of nitric oxide (NO) by RH-infected macrophages was not enhanced. The elimination of CD8+ T cells from splenic cells significantly reduced their inhibitory action on parasite proliferation as well as their cytotoxic activity against RH-infected macrophages, but it did not affect the production of IFN-gamma. Treatment of CD8+ T-enriched splenic cells from the immunized mice with concanamycin A, but not an anti-Fas ligand monoclonal antibody, significantly reduced their anti-proliferative and killing capabilities, suggesting that the CD8+ T cells induced by immunization with RH antigen and live bradyzoites of the Beverley strain may exert protection against T. gondii infection at least in part through granule-dependent cytotoxic activities.     

Persistent parasites and immunologic memory in cutaneous leishmaniasis: implications for vaccine designs and vaccination strategies

Despite a plethora of publications on the murine model of cutaneous leishmaniasis and their contribution to our understanding of the factors that regulate the development of CD4+ T cell immunity in vivo, there is still no effective vaccine against the human disease. While recovery from natural or experimental infection with Leishmania major, the causative agent of human cutaneous leishmaniasis, results in persistence of parasites at the primary infection site and the development of long-lasting immunity to reinfection, vaccination with killed parasites or recombinant proteins induces only short-term protection. The reasons for the difference in protective immunity following recovery from live infection and vaccination with heat-killed parasites are not known. This may in part be related to persistence of live parasites following healing of primary cutaneous lesions, because complete clearance of parasites leads to rapid loss of infection-induced immunity. Recent reports indicate that in addition to persistent parasites, IL-10-producing natural regulatory T cells may also play critical roles in the maintenance and loss of infection-induced immunity. This review focuses on current understanding of the factors that regulate the development, maintenance and loss of anti-Leishmania memory responses and highlights the role of persistent parasites and regulatory T cells in this process. Understanding these factors is crucial for designing effective vaccines and vaccination strategies against cutaneous leishmaniasis.     

The role of T cells in pathogenesis and protective immunity to murine malaria.

T-cell-mediated immunity to a virulent strain of Plasmodium berghei NK65 (Pb NK65) and to an attenuated derivative (Pb XAT) of the strain were examined in CBA mice by the administration of monoclonal antibodies against T-cell subsets or interferon-gamma (IFN-gamma). The injection of anti-CD8+ or anti-IFN-gamma delayed the mortality of mice infected with Pb NK65, although it did not affect the parasitaemia. In the late stage of PB NK65 infection, T cells, especially CD8+ T cells, were increased in number in the liver at the expense of splenic CD8+ T cells. These CD8+ T cells released IFN-gamma in culture without antigen stimulation and were thought to induce tumour necrosis factor-alpha (TNF-alpha) production by the cells in the liver. In mice infected with Pb XAT, or mice primed with Pb XAT and then challenged with Pb NK65, CD4+ T cells had a crucial role in preventing parasite growth and in protective immunity. IFN-gamma was again the key molecule in protective immunity. These results suggest that T cells stimulated with malaria antigen play important roles both in protective immunity and pathogenesis depending upon their subsets; CD8+ T cells in pathogenesis, and CD4+ T cells in protective immunity. These apparently contradictory responses may be mediated by the same cytokine, IFN-gamma.     

Heterologous immunity in the absence of variant-specific antibodies after exposure to subpatent infection with blood-stage malaria

We examined immunity induced by subpatent blood-stage malaria (undetectable by microscopy) using the rodent malaria parasite, Plasmodium chabaudi chabaudi, postulating that limited infection may allow expansion of antigen-specific T cells that are normally deleted by apoptosis. After three infections drug cured at 48 h, mice were protected against high-dose challenge with homologous or heterologous parasites (different strain or variant). Immunity differed from that generated by three untreated, patent infections. Subpatently infected mice lacked immunoglobulin G (IgG) to variant surface antigens, despite producing similar titers of total malaria-specific IgG to those produced by patently infected mice, including antibodies specific for merozoite surface antigens conserved between heterologous strains. Antigen-specific proliferation of splenocytes harvested prechallenge was significantly higher in subpatently infected mice than in patently infected or naive mice. In subpatently infected mice, lymphoproliferation was similar in response to homologous and heterologous parasites, suggesting that antigenic targets of cell-mediated immunity were conserved. A Th1 cytokine response was evident during challenge. Apoptosis of CD4+ and CD8+ splenic lymphocytes occurred during patent but not subpatent infection, suggesting a reason for the relative prominence of cell-mediated immunity after subpatent infection. In conclusion, subpatent infection with blood stage malaria parasites induced protective immunity, which differed from that induced by patent infection and targeted conserved antigens. These findings suggest that alternative vaccine strategies based on delivery of multiple parasite antigens at low dose may induce effective immunity targeting conserved determinants.     

Genetically attenuated Plasmodium berghei liver stages persist and elicit sterile protection primarily via CD8 T cells

Live-attenuated Plasmodium liver stages remain the only experimental model that confers complete sterile protection against malaria. Irradiation-attenuated Plasmodium parasites mediate protection primarily by CD8 T cells. In contrast, it is unknown how genetically attenuated liver stage parasites provide protection. Here, we show that immunization with uis3(-) sporozoites does not cause breakthrough infection in T and B-cell-deficient rag1(-/-) and IFN-gamma(-/-) mice. However, protection was abolished in these animals, suggesting a crucial role for adaptive immune responses and interferon-gamma. Although uis3(-) immunization induced Plasmodium-specific antibodies, B- cell-deficient mice immunized with uis3(-) sporozoites were completely protected against wild-type sporozoite challenge infection. T-cell depletion experiments before parasite challenge showed that protection is primarily mediated by CD8 T cells. In good agreement, adoptive transfer of total spleen cells and enriched CD8 T cells from immunized animals conferred sterile protection against malaria transmission to recipient mice, whereas adoptive transfer of CD4 T cells was less protective. Importantly, primaquine treatment completely abolished the uis3(-)-mediated protection, indicating that persistence of uis3(-)-attenuated liver stages is crucial for their protective action. These findings establish the basic immune mechanisms underlying protection induced by genetically attenuated Plasmodium parasites and substantiate their use as vaccines against malaria.     

Specific inflammatory cell infiltration of hepatic schizonts in BALB/c mice immunized with attenuated Plasmodium yoelii sporozoites.

We compared immunization of BALB/c mice with radiation-attenuated versus killed sporozoites of the rodent malaria parasite, Plasmodium yoelii. We employed a suboptimal schedule of only two immunizations, in expectation that some parasites might break through the resultant low level immunity and that it might thus be possible to study the response of the host against these 'breakthrough' schizonts. As a measure of protective immunity, we used histological means to determine the percentages of challenge sporozoites prevented from completing development into hepatic schizonts within the liver. Immunization with attenuated sporozoites led to almost complete protection, whereas immunization with similar dosages of killed sporozoites led to approximately a 75% protection. Fluorescent antibody titers against sporozoites were similar in both sets of immunized animals. However, serum from mice immunized with attenuated sporozoites had a protective effect upon passive transfer into immunologically naive mice subsequently challenged with normal sporozoites; serum from mice immunized with killed sporozoites had no such effect. When mice suboptimally immunized with attenuated sporozoites were challenged, we observed breakthrough schizonts being infiltrated with inflammatory cells, primarily mononuclear cells, and neutrophils; partial depletion of CD4+ or CD8+ cells within these mice prior to challenge prevented the infiltration of breakthrough schizonts. Thus, cellular infiltration of schizonts was apparently secondary to earlier action by lymphocytes. This infiltration was also not observed in mice immunized with killed sporozoites. The more effective protective immunity induced by attenuated sporozoites could be due to their ability to release antigen into the cytoplasm of hepatocytes that they invade or their ability to continue differentiating, thereby presenting new antigens that are not seen after immunization with killed sporozoites.     

CD4-mediated and CD8-mediated cytotoxic and proliferative immune responses to Toxoplasma gondii in seropositive humans.

Both CD4+ and CD8+ cytotoxic T lymphocytes (CTL) are part of the human immune response to Toxoplasma gondii infection. To further our understanding of Toxoplasma immunity, we investigated factors influencing stimulation of CD4+ or CD8+ human T. gondii-specific immune cells. Both antigen-pulsed and Toxoplasma-infected antigen-presenting cells (APC) induced cell proliferation. Toxoplasma-infected APC elicited strong proliferation of CD4+ cells, but little or no proliferation of CD8+ cells, unless high antigen loads were used. Toxoplasma-infected APC stimulated specific cytotoxicity poorly or not at all, owing to death of stimulated cultures, whereas antigen-pulsed APC strongly elicited specific cytotoxicity. Cytotoxicity elicited by either type of APC resided exclusively in CD4+ T cells in polyclonal cultures. Thus, Toxoplasma-infected APC elicited stronger CD4-mediated than CD8-mediated cell proliferation and generated CD4+ CTL more readily than CD8+ CTL. Nonetheless, specific CD8+ memory cells were demonstrated, and rare CD8+ Toxoplasma-specific CTL were subcloned. Fixed Toxoplasma-infected APC (which induce CD8+ CTL) also elicited cell proliferation, but polyclonal cultures stimulated with these infected APC did not die. Unfixed Toxoplasma-infected APC strongly inhibited phytohemagglutinin-induced cell proliferation, whereas fixed APC did not. These data suggested that infected APC were inhibitory or lethal to some immune cells. Further investigations into interactions between immune cells and Toxoplasma-infected cells likely will help elucidate factors involved in the immunopathogenesis of Toxoplasma infection. As other intracellular parasites, including Plasmodium spp. and Leishmania spp., also elicit CD4+ CTL, such work may help establish paradigms governing immunity to intracellular parasites.     

Resistance of regulatory T cells to glucocorticoid-induced [corrected] TNFR family-related protein (GITR) during Plasmodium yoelii infection

CD4+ T cells are the major effector T cells against blood-stage Plasmodium yoelii infection. On the other hand, the lethal strain of P. yoelii (PyL) has acquired an escape mechanism from host T cell immunity by activating CD4+CD25+ regulatory T cells (Treg). Although the activation of Treg during PyL infection precludes the clearance of PyL from mice, it remains unclear whether activation of Treg is attributable to a specific response against PyL infection. Thus, we examined here whether Treg proliferate in an antigen-dependent manner during PyL infection. We also investigated the effector and regulatory mechanisms of Treg. Infection with PyL increased the number of CD4+CD25+ T cells, in which expression of Foxp3 mRNA is up-regulated. The Treg that were transferred into mice infected with PyL, but not with a non-lethal strain of P. yoelii (PyNL), proliferated during the initial 5 days following infection. The Treg from PyL-infected mice showed strong suppression compared with those from naive or PyNL-infected mice, and could suppress T cell activation by recognizing PyL- but not PyNL-derived antigens. Furthermore, the suppressive function of Treg activated in PyL-infected but not in naive mice could not be inhibited by treatment with an anti-glucocorticoid-induced TNFR family-related protein (GITR) mAb. These findings indicate that PyL infection specifically activates Treg that are specific for PyL-derived antigens. The infection also induces resistance for Treg to GITR signaling, and this eventually contributes to the escape of parasites from host T cell immunity.     

Evidence for a significant role of CD4+ T cells in adoptive immunity to Listeria monocytogenes in the liver.

Although the ability of CD8+ T cells to adoptively immunize mice against Listeria monocytogenes in the spleen is well established, the role of different T-cell subsets in anti-bacterial protection in the liver, a major target of Listeria infection, remains unclear. Therefore, the ability of sorted CD4+ and CD8+ T cells to adoptively immunize mice against a L. monocytogenes infection in the liver was studied. The results show that positively sorted CD4+ T cells from day 7 Listeria-immune mice were as effective as sorted CD8+ cells in transferring significant anti-Listeria protection in the liver. Similar findings were obtained when CD4+ and CD8+ T cells, negatively selected by antibody-induced complement-mediated depletion in vitro, were used for adoptive transfer. CD8+ T cells, however, were more efficient than CD4+ T cells in transferring protection in the spleen. Taken together, the results show that CD4+ T cells are at least as protective as CD8+ T cells against a L. monocytogenes infection in the liver, thereby arguing against the view that CD4+ T cells are of limited importance in adoptive immunity against listeriosis.     

Inhibition of nitric oxide interrupts the accumulation of CD8+ T cells surrounding Plasmodium berghei-infected hepatocytes.

The elimination of liver-stage malaria parasites by nitric oxide (NO)-producing hepatocytes is regulated by T cells. Both CD8+ and CD4+ T cells, which surround infected hepatocytes, are evident by 24 h after sporozoite challenge in Brown Norway rats previously immunized with irradiated Plasmodium berghei sporozoites. While the number of CD4+ T cells remained the same beyond 24 h postchallenge, the number of CD8+ T cells increased three- and sixfold by 31 and 44 h, respectively. This increase in the number of CD8+ T cells correlated with a decrease in the number of intrahepatic parasites. In immunized rats, intrahepatic parasites were reduced in number by 31 h after sporozoite challenge and cleared from the liver by 44 h, as visualized by P. berghei-specific DNA in situ hybridization. If immunized rats were treated with aminoguanidine, a substrate inhibitor of NO synthase, at the time of challenge, liver-stage protection was blocked, as shown by the increase in parasite liver burden. Further histological examination of infected livers from immunized animals treated with aminoguanidine revealed fewer and smaller cellular infiltrates surrounding the infected hepatocytes, and the number of CD8+ T cells that normally accumulate within the infiltrates was drastically reduced. Consequently, the infected hepatocytes were not cleared from the liver. We hypothesize that the early production of NO may promote the influx and/or enhance local proliferation of malaria parasite-specific CD8+ T cells or a CD8+ T-cell subset which is required for parasite clearance.     

Memory phenotype CD8(+) T cells persist in livers of mice protected against malaria by immunization with attenuated Plasmodium berghei sporozoites

Natural exposure to Plasmodium parasites induces short-lived protective immunity. In contrast, exposure to radiation-attenuated sporozoites (gamma spz) promotes long-lasting protection that is in part mediated by CD8(+) T cells that target exoerythrocytic stage antigens. The mechanisms underlying the maintenance of long-lasting protection are currently unclear. The liver is a repository of Plasmodium antigens and may support the development and / or homing of memory T cells. While activated CD8(+) T cells are presumed to die in the liver, the fate of anti-Plasmodium CD8(+) T cells remains unknown. We propose that inflammatory conditions in the liver caused by Plasmodium parasites may allow some effector CD8(+) T cells to survive and develop into memory cells. To support this hypothesis, in this initial study we demonstrate that liver mononuclear cells from P. berghei gamma spz-immune mice transferred protection to naive recipients and moreover, that CD4(+) and CD8(+) T cells responded to Plasmodium antigens by up-regulating activation / memory markers. While CD4(+) T cells under went a transient activation following immunization with gamma spz, CD8(+) T cells expanded robustly after spz challenge and exhibited stable expression of CD44(hi) and CD45RB(lo) during protracted protection. These results establish a key role for intrahepatic T cells in long-lasting protection against malaria.     

Cell-mediated immunity to the asexual blood stages of malarial parasites: animal models

Studies using experimental models of malaria in immunodeficient mice and chickens have shown that resistance to blood-stage infection is mediated by protective antibodies and T cell-dependent cell-mediated mechanisms of immunity. Depending on the infecting species of Plasmodium and prior experience of the host, either humoral or cell-mediated immune mechanisms predominate. Cell-mediated immunity has been adoptively transferred with CD4+ splenic T cells, and with antigen-specific T cell lines and clones. Since ascending parasitemia occurs in all instances, the transferred cells do not kill plasmodia directly but appear to activate effector mechanisms capable of destroying the invading parasites. Both CD4+ and CD8+ T cells were found to increase in the spleens of malarious mice. Depletion of CD4+ T cells prevented nude mice adoptively transferred with immune splenic T cells from clearing parasitemia. In contrast, late treatment with anti-CD4 antibody had little if any effect. The converse was true when anti-CD8 antibody was utilized, i.e., a significant number of mice treated with anti-CD8 antibody after parasitemia became patent and had difficulty clearing blood parasites. These data suggest that during infection CD8+ T cells may become activated by CD4+ T cells responding to malarial antigens. These CD8+ T cells may be directly cytotoxic or secrete additional cytokines thereby amplifying the role of CD4+ T cells in the activation of anti-parasite effector mechanisms. Finally, a hypothesis is presented to explain how the parasite in natural infections may activate T cell-dependent effector mechanisms in order to control its numbers in host tissues thereby ensuring the survival of both parasite and host.     

Lymphocyte phenotypes in the intestinal mucosa of sheep infected with Trichostrongylus colubriformis

Monoclonal antibodies to ovine lymphocyte surface antigens were used in an immunohistochemical study of the intestine of sheep. In the epithelium CD8+ cells predominated whereas the majority of lamina propria T lymphocytes were CD4+. Infection of sheep with the parasitic nematode Trichostrongylus colubriformis including sufficiently large numbers of parasites to induce protective immunity did not alter the number of CD4+ or CD8+ lymphocytes in the intestinal mucosa. In contrast, exposure of naive sheep to a single large infection of T. colubriformis resulted in a substantial decrease in number of CD8+ cells and moderate decreases in number of CD4+ cells in the duodenal but not the jejunal mucosa. MHC class II antigens were not detected either in or on epithelial cells of the sheep small intestine.     

The role of intrahepatic lymphocytes in mediating protective immunity induced by attenuated Plasmodium berghei sporozoites

Summary: Exposure to irradiated Plasmodium sporozoites (g‐spz) results in protection against malaria. Like infectious spz, g‐spz colonize hepatocytes to undergo maturation. Disruption of liver stage development prevents the generation of protection, which appears, therefore, to depend on liver stage antigens. Although some mechanisms of protection have been identified, they do not include a role for intrahepatic mononuclear cells (IHMC). We demonstrated that P. berghei g‐spz‐immune murine IHMC adoptively transfer protection to naive recipients. Characterization of intrahepatic CD4+ T cells revealed an immediate, albeit transient, response to g‐spz, while the response of CD8+ T cells is delayed until acquisition of protection. It is presumed that activated CD8+ T cells home to the liver to die; g‐spz‐induced CD8+CD45RBloCD44hi T cells, however, persist in the liver, but not the spleen, during protracted protection. The association between CD8+CD45RBloCD44hi T cells and protection has been verified using MHC class I and CD1 knockout mice and mice with disrupted liver stage parasites. Based on kinetic studies, we propose that interferon‐g, presumably released by intrahepatic effector CD8+ T cells, mediates protection; the persistence of CD8+ T cells is, in turn, linked to Plasmodium antigen depots and cytokines released by CD4+ T cells and/or NK T cells.     

DNA sequences encoding CD4+ and CD8+ T-cell epitopes are important for efficient protective immunity induced by DNA vaccination with a Trypanosoma cruzi gene

Immunization of BALB/c mice with a plasmid containing the gene for Trypanosoma cruzi trans-sialidase (TS) induced antibodies that inhibited TS enzymatic activity, CD4+ Th1 and CD8+ Tc1 cells, and protective immunity against infection. We used this model to obtain basic information on the requirement of CD4 or CD8 or B-cell epitopes for an effective DNA-induced immunity against T. cruzi infection. For that purpose, mice were immunized with plasmids containing DNA sequences encoding (i) the entire TS protein, (ii) the TS enzymatic domain, (iii) the TS CD4+ T-cell epitopes, (iv) the TS CD8+ T-cell epitope, or (v) TS CD4+ and CD8+ T-cell epitopes. Plasmids expressing the entire TS or its enzymatic domain elicited similar levels of TS-inhibitory antibodies, gamma interferon (IFN-gamma)-producing T cells, and protective immunity against infection. Although the plasmid expressing TS CD4 epitopes was immunogenic, its protective efficacy against experimental infection was limited. The plasmid expressing the CD8 epitope was poorly immunogenic and provided little protective immunity. The reason for the limited priming of CD8+ T cells was due to a requirement for CD4+ T cells. To circumvent this problem, a plasmid expressing both CD4+ and CD8+ T-cell epitopes was produced. This plasmid generated levels of IFN-gamma-producing T cells and protective immunity comparable to that of the plasmid expressing the entire catalytic domain of TS. Our observations suggest that plasmids expressing epitopes recognized by CD4+ and CD8+ T cells may have a better protective potential against infection with T. cruzi.