Induction of protective immunity against malaria by priming-boosting immunization with recombinant cold-adapted influenza and modified vaccinia Ankara viruses expressing a CD8+-T-cell epitope derived from the circumsporozoite protein of Plasmodium yoelii

We immunized mice with an attenuated (cold-adapted) influenza virus followed by an attenuated vaccinia virus (modified vaccinia virus Ankara), both expressing a CD8(+)-T-cell epitope derived from malaria sporozoites. This vaccination regimen elicited high levels of protection against malaria. This is the first time that the vaccine efficacy of a recombinant cold-adapted influenza virus vector expressing a foreign antigen has been evaluated.     

Naturally acquired CD8+ cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein.

In rodent malaria model systems, protective immunity induced by immunization with irradiated sporozoites is eliminated by in vivo depletion of CD8+ T cells, and adoptive transfer of CTL clones against the circumsporozoite protein protects against malaria. We recently demonstrated that volunteers immunized with irradiated Plasmodium falciparum sporozoites produce CTL against peptide 368-390 of the P. falciparum circumsporozoite protein. To determine whether natural exposure to malaria induced similar CTL, we studied 11 adult, male, life-long residents of a highly malarious area of Kenya, who were selected because their lymphocytes had been shown to proliferate after stimulation with peptides 361-380, 371-390, or 368-390 and because nine had been resistant to malaria in previous studies. In four of the 11 individuals there was peptide-specific, genetically restricted, CTL activity. In all four individuals, this activity was unaffected by depletion of CD4+ T cells. In three volunteers the activity was eliminated or reduced by depletion of CD8+ T cells; in the fourth volunteer the CD8+ T cell depletion was uninterpretable. This first demonstration of CD8+ T cell, genetically restricted, Ag-specific CTL against a malaria protein among individuals exposed to endemic malaria provides a foundation for studying the relationship between circulating CTL and resistance to malaria infection.     

Synergistic role of CD4+ and CD8+ T lymphocytes in IFN-gamma production and protective immunity induced by an attenuated Toxoplasma gondii vaccine.

BALB/c mice vaccinated with a temperature-sensitive mutant (TS-4) of Toxoplasma gondii develop complete resistance to lethal challenge with a highly virulent toxoplasma strain (RH). This immunity is known to be dependent on IFN-gamma synthesis. In vitro and in vivo T cell depletions were performed in order to identify the subsets responsible for both protective immunity and IFN-gamma production. When stimulated with crude tachyzoite Ag in vitro, CD4+ cells from vaccinated mice produced high levels of TH1 cytokines (IL-2 and IFN-gamma) but not TH2 cytokines (IL-4 and IL-5). CD8+ cells, in contrast, produced less IFN-gamma and no detectable IL-2. Nevertheless, they could be induced to synthesize IFN-gamma when exposed in culture to exogenous IL-2. In vivo treatment with anti-CD4 plus anti-CD8 or anti-IFN-gamma antibodies during challenge infection completely abrogated resistance to T. gondii. In contrast, treatment with anti-CD4 alone failed to reduce immunity, whereas anti-CD8 treatment partially decreased vaccine-induced resistance. These results suggest that although IFN-gamma and IL-2-producing CD4+ lymphocytes are induced by vaccination, IFN-gamma-producing CD8+ T cells are the major effectors of immunity in vivo. Nevertheless, CD4+ lymphocytes appear to play a synergistic role in vaccine-induced immunity, probably through the augmentation of IFN-gamma synthesis by the CD8+ effector cells. This hypothesis is supported by the observation that when giving during vaccination, as opposed to after challenge, anti-CD4 antibodies are capable of blocking protective immunity.     

IL-12 and NK cells are required for antigen-specific adaptive immunity against malaria initiated by CD8+ T cells in the Plasmodium yoelii model.

CD8+ T cells have been implicated as critical effector cells in protection against preerythrocytic stage malaria, including the potent protective immunity of mice and humans induced by immunization with radiation-attenuated Plasmodium spp. sporozoites. This immunity is directed against the Plasmodium spp. parasite developing within the host hepatocyte and for a number of years has been presumed to be mediated directly by CD8+ CTL or indirectly by IFN-gamma released from CD8+ T cells. In this paper, in BALB/c mice, we establish that after immunization with irradiated sporozoites or DNA vaccines parasite-specific CD8+ T cells trigger a novel mechanism of adaptive immunity that is dependent on T cell- and non-T cell-derived cytokines, in particular IFN-gamma and IL-12, and requires NK cells but not CD4+ T cells. The absolute requirement for CD8+ T cells to initiate such an effector mechanism, and the requirement for IL-12 and NK cells in such vaccine-induced protective immunity, are unique and underscore the complexity of the immune responses that protect against malaria and other intracellular pathogens.     

Adoptive transfer of CD8+ T cells from immune animals does not transfer immunity to blood stage Plasmodium yoelii malaria

The malaria parasite, Plasmodium yoelii 17X, causes a self-limited, nonlethal infection characterized, in the blood stage, by preferential invasion of reticulocytes. Previous studies have suggested that immunity to the blood stage infection may be related to enhanced levels of class I MHC Ag on the parasitized reticulocyte surface and can be adoptively transferred to immunodeficient mice by immune CD8+ T cells in the absence of CD4+ T cells. To further examine the mechanisms of CD8+ T cell involvement in immunity to blood stage P. yoelii infection, we performed in vivo CD8 depletion and adoptive transfer experiments. Depletion of CD8+ T cells during primary blood stage infection in BALB/c mice did not diminish the ability of the mice to resolve their infections. Spleen cells from immune BALB/c and C57BL/10 mice were transferred to BALB/c-nu/nu and C57BL/10-nu/nu mice, respectively. The recipient mice were CD4 depleted in vivo to kill any transferred CD4+ T cells. The mice failed to control the infection. Populations of CD4-, CD8+ T cells were transferred from immune CBA/CaJ donors to in vivo CD4-depleted CBA/CaJ recipients. The mice were unable to control the infection. Although immune unfractionated spleen cells transferred rapid protection in all three mouse strains and immune CD4+ T cells transferred immunity in the two mouse strains studied, CD8+ T cells by themselves were neither protective nor did they enhance immunity.     

A T cell clone directed at the circumsporozoite protein which protects mice against both Plasmodium yoelii and Plasmodium berghei.

Clone B is a cytotoxic T cell clone induced by immunization with Plasmodium yoelii sporozoites which recognizes an epitope on both the P. yoelii and Plasmodium berghei circumsporozoite proteins. It is CD8, uses the V beta 8.1 TCR, and is Kd restricted. When adoptively transferred, it protects mice against infection by both species of malaria sporozoites, and this protection is dependent on IFN-gamma. Clone B cells are more broadly reactive and protective than previously described murine T cell clones against malaria. Clone B may be an important model for immune protection against the spectrum of variant parasites in nature.     

Protective immunity induced with malaria vaccine, RTS,S, is linked to Plasmodium falciparum circumsporozoite protein-specific CD4+ and CD8+ T cells producing IFN-gamma

The Plasmodium falciparum circumsporozoite (CS) protein-based pre-erythrocytic stage vaccine, RTS,S, induces a high level of protection against experimental sporozoite challenge. The immune mechanisms that constitute protection are only partially understood, but are presumed to rely on Abs and T cell responses. In the present study we compared CS protein peptide-recalled IFN-gamma reactivity of pre- and RTS,S-immune lymphocytes from 20 subjects vaccinated with RTS,S. We observed elevated IFN-gamma in subjects protected by RTS,S; moreover, both CD4(+) and CD8(+) T cells produced IFN-gamma in response to CS protein peptides. Significantly, protracted protection, albeit observed only in two of seven subjects, was associated with sustained IFN-gamma response. This is the first study demonstrating correlation in a controlled Plasmodia sporozoite challenge study between protection induced by a recombinant malaria vaccine and Ag-specific T cell responses. Field-based malaria vaccine studies are in progress to validate the establishment of this cellular response as a possible in vitro correlate of protective immunity to exo-erythrocytic stage malaria vaccines.     

A purified parasite antigen (p30) mediates CD8+ T cell immunity against fatal Toxoplasma gondii infection in mice

Induction of protective immunity against acute and chronic toxoplasmosis can be achieved using p30, the major membrane and excreted/secreted protein of Toxoplasma gondii. This protein, when administered to outbred mice in the presence of the saponin Quil A, is able to induce almost 100% protection against acute infection without evidence of intracerebral cyst development. Adoptive transfer of immune splenocytes from immunized inbred A/J mice conferred a significant level (p less than 0.001) of protection against subsequent challenge. Phenotypic analysis in outbred as well as two different strains of inbred mice (A/J and C57BL/6) demonstrated that CD8+ T cells are selectively stimulated by this immunization protocol. T cell depletion studies using specific mAb directed at either CD3+ or CD8+ T cell phenotype, followed by adoptive transfer, failed to confer protective immunity, whereas CD4+ depletion had no effect. These cytotoxic CD8+ T cells produced high titers of both IFN-gamma and IL-2. Moreover, these CD8+ T cells were directly parasiticidal against radiolabeled extracellular T. gondii, further supporting the critical immune function of these p30 Ag-specific CD8+ T cells in host immunity against T. gondii infection.     

Foxp3+ regulatory T cells impede the priming of protective CD8+ T cells

T cell activation is controlled by incompletely defined opposing stimulation and suppression signals that together sustain the balance between optimal host defense against infection and peripheral tolerance. In this article, we explore the impacts of Foxp3(+) regulatory T cell (Treg) suppression in priming Ag-specific T cell activation under conditions of noninfection and infection. We find the transient ablation of Foxp3(+) Tregs unleashes the robust expansion and activation of peptide-stimulated CD8(+) T cells that provide protection against Listeria monocytogenes infection in an Ag-specific fashion. By contrast, Treg ablation had nonsignificant impacts on the CD8(+) T cell response primed by infection with recombinant L. monocytogenes. Similarly, nonrecombinant L. monocytogenes administered with peptide stimulated the expansion and activation of CD8(+) T cells that paralleled the response primed by Treg ablation. Interestingly, these adjuvant properties of L. monocytogenes did not require CD8(+) T cell stimulation by IL-12 produced in response to infection, but instead were associated with sharp reductions in Foxp3(+) Treg suppressive potency. Therefore, Foxp3(+) Tregs impose critical barriers that, when overcome naturally during infection or artificially with ablation, allow the priming of protective Ag-specific CD8(+) T cells.     

A CD8+ T cell heptaepitope minigene vaccine induces protective immunity against Chlamydia pneumoniae

An intact T cell compartment and IFN-gamma signaling are required for protective immunity against Chlamydia. In the mouse model of Chlamydia pneumoniae (Cpn) infection, this immunity is critically dependent on CD8(+) T cells. Recently we reported that Cpn-infected mice generate an MHC class I-restricted CD8(+) Tc1 response against various Cpn Ags, and that CD8(+) CTL to multiple epitopes inhibit Cpn growth in vitro. Here, we engineered a DNA minigene encoding seven H-2(b)-restricted Cpn CTL epitopes, the universal pan-DR epitope Th epitope, and an endoplasmic reticulum-translocating signal sequence. Immunization of C57BL/6 mice with this construct primed IFN-gamma-producing CD8(+) CTL against all seven CTL epitopes. CD8(+) T cell lines generated to minigene-encoded CTL epitopes secreted IFN-gamma and TNF-alpha and exhibited CTL activity upon recognition of Cpn-infected macrophages. Following intranasal challenge with Cpn, a 3.6 log reduction in mean lung bacterial numbers compared with control animals was obtained. Using a 20-fold increase in the Cpn challenging dose, minigene-vaccinated mice had a 60-fold reduction in lung bacterial loads, compared with controls. Immunization and challenge studies with beta(2)-microglobulin(-/-) mice indicated that the reduction of lung Cpn burdens was mediated by the MHC class I-dependent CD8(+) T cells to minigene-included Cpn CTL epitopes, rather than by pan-DR epitope-specific CD4(+) T cells. This constitutes the first demonstration of significant protection achieved by immunization with a CD8(+) T cell epitope-based DNA construct in a bacterial system and provides the basis for the optimal design of multicomponent anti-Cpn vaccines for humans.     

Plasmodium berghei-infected primary hepatocytes process and present the circumsporozoite protein to specific CD8+ T cells in vitro

A substantial and protective response against malaria liver stages is directed against the circumsporozoite protein (CSP) and involves induction of CD8(+) T cells and production of IFN-gamma. CSP-derived peptides have been shown to be presented on the surface of infected hepatocytes in the context of MHC class I molecules. However, little is known about how the CSP and other sporozoite Ags are processed and presented to CD8(+) T cells. We investigated how primary hepatocytes from BALB/c mice process the CSP of Plasmodium berghei after live sporozoite infection and present CSP-derived peptides to specific H-2K(d)-restricted CD8(+) T cells in vitro. Using both wild-type and spect(-/-) P. berghei sporozoites, we show that both infected and traversed primary hepatocytes process and present the CSP. The processing and presentation pathway was found to involve the proteasome, Ag transport through a postendoplasmic reticulum compartment, and aspartic proteases. Thus, it can be hypothesized that infected hepatocytes can contribute in vivo to the elicitation and expansion of a T cell response.     

CD8+ T lymphocytes protective against malaria liver stages are primed in skin-draining lymph nodes

The success of immunization with irradiated sporozoites is unparalleled among the current vaccination approaches against malaria, but its mechanistic underpinnings have yet to be fully elucidated. Using a model mimicking natural infection by Plasmodium yoelii, we delineated early events governing the development of protective CD8(+) T-cell responses to the circumsporozoite protein. We demonstrate that dendritic cells in cutaneous lymph nodes prime the first cohort of CD8(+) T cells after an infectious mosquito bite. Ablation of these lymphoid sites greatly impairs subsequent development of protective immunity. Activated CD8(+) T cells then travel to systemic sites, including the liver, in a sphingosine-1-phosphate (S1P)-dependent fashion. These effector cells, however, no longer require bone marrow-derived antigen-presenting cells for protection; instead, they recognize antigen on parenchymal cells-presumably parasitized hepatocytes. Therefore, we report an unexpected dichotomy in the tissue restriction of host responses during the development and execution of protective immunity to Plasmodium.     

CD8+ CTLs are essential for protective immunity against Encephalitozoon cuniculi infection

Encephalitozoon cuniculi is a protozoan parasite that has been implicated recently as a cause of opportunistic infection in immunocompromised individuals. Protective immunity in the normal host is T cell-dependent. In the present study, the role of individual T cell subtypes in immunity against this parasite has been studied using gene knockout mice. Whereas CD4-/- animals resolved the infection, mice lacking CD8+ T cells or perforin gene succumbed to parasite challenge. The data obtained in these studies suggest that E. cuniculi infection induces a strong and early CD8+ T response that is important for host protection. The CD8+ T cell-mediated protection depends upon the CTL activity of this cell subset, as the host is rendered susceptible to infection in the absence of this function. This is the first report in which a strong dependence upon the cytolytic activity of host CD8+ T cells has been shown to be important in a parasite infection.     

Induction and maintenance of protective CD8+ T cells against malaria liver stages: implications for vaccine development

CD8+ T cells against malaria liver stages represent a major protective immune mechanism against infection. Following induction in the peripheral lymph nodes by dendritic cells (DCs), these CD8+ T cells migrate to the liver and eliminate parasite infected hepatocytes. The processing and presentation of sporozoite antigen requires TAP mediated transport of major histocompatibility complex class I epitopes to the endoplasmic reticulum. Importantly, in DCs this process is also dependent on endosome-mediated cross presentation while this mechanism is not required for epitope presentation on hepatocytes. Protective CD8+ T cell responses are strongly dependent on the presence of CD4+ T cells and the capacity of sporozoite antigen to persist for a prolonged period of time. While human trials with subunit vaccines capable of inducing antibodies and CD4+ T cell responses have yielded encouraging results, an effective anti-malaria vaccine will likely require vaccine constructs designed to induce protective CD8+ T cells against malaria liver stages.     

Imaging effector functions of human cytotoxic CD4+ T cells specific for Plasmodium falciparum circumsporozoite protein

Malaria vaccines, comprised of irradiated Plasmodium falciparum sporozoites or a synthetic peptide containing T and B cell epitopes of the circumsporozoite protein (CSP), elicit multifunctional cytotoxic and non-cytotoxic CD4⁺ T cells in immunised volunteers. Both lytic and non-lytic CD4⁺T cell clones recognised a series of overlapping epitopes within a ‘universal’ T cell epitope EYLNKIQNSLSTEWSPCSVT of CSP (NF54 isolate) that was presented in the context of multiple DR molecules. Lytic activity directly correlated with T cell receptor (TCR) functional avidity as measured by stimulation indices and recognition of naturally occurring variant peptides. CD4⁺ T cell-mediated cytotoxicity was contact-dependent and did not require de novo synthesis of cytotoxic mediators, suggesting a granule-mediated mechanism. Live cell imaging of the interaction of effector and target cells demonstrated that CD4⁺ cytotoxic T cells recognise target cells with their leading edge, reorient their cytotoxic granules towards the zone of contact, and form a stable immunological synapse. CTL attacks induced chromatin condensation, nuclear fragmentation and formation of apoptotic bodies in target cells. Together, these findings suggest that CD4⁺ CTLs trigger target cell apoptosis via classical perforin/granzyme-mediated cytotoxicity, similar to CD8⁺ CTLs, and these multifunctional sporozoite- and peptide-induced CD4⁺ T cells have the potential to play a direct role as effector cells in targeting the exoerythrocytic forms within the liver.     

T9 glioma cells expressing membrane-macrophage colony stimulating factor produce CD4+ T cell-associated protective immunity against T9 intracranial gliomas and systemic immunity against different syngeneic gliomas

Cloned T9 glioma cells (T9-C2) expressing the membrane form of macrophage colony stimulating factor (mM-CSF) inoculated subcutaneously into rats do not grow and glioma-specific immunity is stimulated. Immunotherapy experiments showed that intracranial T9 tumors present for one to four days could be successfully eradicated by peripheral vaccination with T9-C2 cells. CD4+ and CD8+ T splenocytes from immunized rats, when restimulated in vitro with T9 cells, produced interleukin-2 and -4. Protective immunity against intracranial T9 gliomas could only be adoptively transferred into naive rats by the CD4+ splenocytes obtained from T9-C2 immunized rats. Rats immunized by the T9-C2 tumor cells also resisted two different syngeneic gliomas (RT2 and F98) but allowed a syngeneic NUTU-19 ovarian cancer to grow. Such cross-protective immunity against unrelated gliomas suggests that mM-CSF transfected tumor cells have immunotherapeutic potential for use as an allogeneic tumor vaccine.     

NKT cells provide help for dendritic cell-dependent priming of MHC class I-restricted CD8+ T cells in vivo

Dendritic cells (DC) are potent APCs for naive T cells in vivo. This is evident by inducing T cell responses through adoptive DC transfer. Priming specific CTL responses in vivo often requires "help". We study alternative sources of help in DC-dependent priming of MHC class I-restricted CTL. Priming an anti-viral CTL response in naive B6 mice by adoptive transfer of antigenic peptide-pulsed DC required CD4(+) T cell help. CTL priming was facilitated by providing MHC class II-dependent specific help. Furthermore, transfers of MHC class II-deficient pulsed DC into naive, normal hosts, or DC transfers into naive, CD4(+) T cell-depleted hosts primed CTL inefficiently. Pretreatment of DC with immune-stimulating oligodeoxynucleotides rendered them more efficient for CD4(+) T cell-independent priming of CTL. DC copresenting a K(b)-binding antigenic peptide and the CD1d-binding glycolipid alpha-galactosyl-ceramide efficiently primed CTL in a class II-independent way. To obtain NKT cell-dependent help in CTL priming, the same DC had to present both the peptide and the glycolipid. CTL priming by adoptive DC transfer was largely NK cell-dependent. The requirement for NK cells was only partially overcome by recruiting NKT cell help into DC-dependent CTL priming. NKT cells thus are potent helper cells for DC-dependent CTL priming.     

CD8+ T cell-dependent elimination of dendritic cells in vivo limits the induction of antitumor immunity

The fate of dendritic cells (DC) after they have initiated a T cell immune response is still undefined. We have monitored the migration of DC labeled with a fluorescent tracer and injected s.c. into naive mice or into mice with an ongoing immune response. DC not loaded with Ag were detected in the draining lymph node in excess of 7 days after injection with maximum numbers detectable approximately 40 h after transfer. In contrast, DC that had been loaded with an MHC class I-binding peptide disappeared from the lymph node with kinetics that parallel the known kinetics of activation of CD8+ T cells to effector function. In the presence of high numbers of specific CTL precursors, as in TCR transgenic mice, DC numbers were significantly decreased by 72 h after injection. The rate of DC disappearance was extremely rapid and efficient in recently immunized mice and was slower in "memory" mice in which memory CD8+ cells needed to reacquire effector function before mediating DC elimination. We also show that CTL-mediated clearance of Ag-loaded DC has a notable effect on immune responses in vivo. Ag-specific CD8+ T cells failed to divide in response to Ag presented on a DC if the DC were targets of a pre-existing CTL response. The induction of antitumor immunity by tumor Ag-loaded DC was also impaired. Therefore, CTL-mediated clearance of Ag-loaded DC may serve as a negative feedback mechanism to limit the activity of DC within the lymph node.     

Toxoplasma gondii: protective immunity against experimental toxoplasmosis induced by a DNA vaccine encoding the perforin-like protein 1

Toxoplasma gondii is an important zoonotic parasite infecting about one third of the world population, causing congenital infections and eye disease. T. gondii perforin-like protein 1 (TgPLP1) is believed to be involved in the acute virulence of T. gondii in mice, and is therefore of interest as a vaccine candidate. In this study, we constructed a DNA vaccine expressing TgPLP1, and evaluated the immune response in Kunming mice. The gene sequence encoding TgPLP1 was inserted into the eukaryotic expression vector pVAX I, and Kunming mice were immunized intramuscularly with the plasmid. After immunization, we evaluated the immune response using lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally with 1 × 10³ tachyzoites of the virulent T. gondii RH strain. The results showed that pVAX/TgPLP1 alone or with pVAX/IL-18 developed specific anti-TLA (T. gondii lysate antigen) antibodies and specific lymphocyte proliferative responses. Co-injection of pVAX/IL-18 significantly increased the production of IFN-γ and IL-2. Further, challenge experiments showed that co-immunization of pVAX/TgPLP1 with pVAX/IL-18 significantly (P < 0.05) increased survival time (12.7 ± 1.2 days) of immunized mice, compared with pVAX/TgPLP1 alone (11.3 ± 0.9 days). These results demonstrate that TgPLP1 is a potential vaccine candidate against toxoplasmosis, worth further evaluation in other animal hosts. IL-18 could enhance the immune effect of TgPLP1, prolonging the survival time of immunized mice.     

Enhanced CD8+ T cell immune responses and protection elicited against Plasmodium berghei malaria by prime boost immunization regimens using a novel attenuated fowlpox virus

Sterile immunity can be provided against the pre-erythrocytic stages of malaria by IFN-gamma-secreting CD8(+) T cells that recognize parasite-infected hepatocytes. In this study, we have investigated the use of attenuated fowlpox virus (FPV) strains as recombinant vaccine vectors for eliciting CD8(+) T cells against Plasmodium berghei. The gene encoding the P. berghei circumsporozoite (PbCS) protein was inserted into an FPV vaccine strain licensed for use in chickens, Webster's FPV, and the novel FPV vaccine strain FP9 by homologous recombination. The novel FP9 strain proved more potent as a vaccine for eliciting CD8(+) T cell responses against the PbCS Ag. Sequential immunization with rFP9 and recombinant modified vaccinia virus Anakara (MVA) encoding the PbCS protein, administered by clinically acceptable routes, elicited potent CD8(+) T cell responses against the PbCS protein. This immunization regimen elicited substantial protection against a stringent liver-stage challenge with P. berghei and was more immunogenic and protective than DNA/MVA prime/boost immunization. However, further improvement was not achieved by sequential (triple) immunization with a DNA vaccine, FP9, and MVA.