Fidel Zavala

In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens.

Cytotoxic T lymphocytes (CTL) respond to antigenic peptides presented on MHC class I molecules. On most cells, these peptides are exclusively of endogenous, cytosolic origin. Bone marrow-derived antigen-presenting cells, however, harbor a unique pathway for MHC I presentation of exogenous antigens. This mechanism permits cross-presentation of pathogen-infected cells and the priming of CTL responses against intracellular microbial infections. Here, we report a novel diphtheria toxin-based system that allows the inducible, short-term ablation of dendritic cells (DC) in vivo. We show that in vivo DC are required to cross-prime CTL precursors. Our results thus define a unique in vivo role of DC, i.e., the sensitization of the immune system for cell-associated antigens. DC-depleted mice fail to mount CTL responses to infection with the intracellular bacterium Listeria monocytogenes and the rodent malaria parasite Plasmodium yoelii.

Quantification of antigen specific CD8+ T cells using an ELISPOT assay

An ELISPOT assay to detect and determine the number of antigen specific CD8+ T cells was standardized using cloned murine CD8+ T cells specific for the epitope SYVPSAEQI of a rodent malaria antigen. This assay is based on the detection of IFN-gamma secretion by single cells after their stimulation with antigen. The interferon secretion is visualized as spots revealed by using enzyme labeled anti-IFN-gamma monoclonal antibodies. Using known numbers of cloned murine CD8+ T cells it was determined that the assay detects 80-95% of these CD8+ T cells. The optimal culture conditions for the stimulation of the CD8+ T cells were determined and the antigen concentration, number of antigen presenting cells and supplement of growth factors required to perform the assay were defined. This ELISPOT assay can be performed with spleen cells from immunized mice, and provide the precise number of antigen specific CD8+ T cells present in mixed lymphocyte populations. This method is more sensitive than the chromium-51 release assay, and much simpler than the conventional precursor frequency analysis, providing the number of antigen specific CD8+ T cells in 36-48 h.

The circumsporozoite protein is an immunodominant protective antigen in irradiated sporozoites

Malaria infection starts when mosquitoes inject sporozoites into the skin. The parasites enter the blood stream and make their way to the liver where they develop into the exo-erythrocytic forms (EEFs). Immunization with irradiated sporozoites (IrSp) leads to robust protection against malaria infection in rodents, monkeys and humans by eliciting antibodies to circumsporozoite protein (CS) that inhibit sporozoite infectivity, and T cells that destroy the EEFs. To study the role of non-CS antigens in protection, we produced CS transgenic mice that were tolerant to CS T-cell epitopes. Here we show that in the absence of T-cell-dependent immune responses to CS, protection induced by immunization with two doses of IrSp was greatly reduced. Thus, although hundreds of other Plasmodium genes are expressed in sporozoites and EEFs, CS is a dominant protective antigen. Nevertheless, sterile immunity could be obtained by immunization of CS transgenics with three doses of IrSp.

Antigen targeting to dendritic cells elicits long-lived T cell help for antibody responses

Resistance to several prevalent infectious diseases requires both cellular and humoral immune responses. T cell immunity is initiated by mature dendritic cells (DCs) in lymphoid organs, whereas humoral responses to most antigens require further collaboration between primed, antigen-specific helper T cells and naive or memory B cells. To determine whether antigens delivered to DCs in lymphoid organs induce T cell help for antibody responses, we targeted a carrier protein, ovalbumin (OVA), to DCs in the presence of a maturation stimulus and assayed for antibodies to a hapten, (4-hydroxy-3-nitrophenyl) acetyl (NP), after boosting with OVA-NP. A single DC-targeted immunization elicited long-lived T cell helper responses to the carrier protein, leading to large numbers of antibody-secreting cells and high titers of high-affinity antihapten immunoglobulin Gs. Small doses of DC-targeted OVA induced higher titers and a broader spectrum of anti-NP antibody isotypes than large doses of OVA in alum adjuvant. Similar results were obtained when the circumsporozoite protein of Plasmodium yoelii was delivered to DCs. We conclude that antigen targeting to DCs combined with a maturation stimulus produces broad-based and long-lived T cell help for humoral immune responses.

Detection of malaria liver-stages in mice infected through the bite of a single Anopheles mosquito using a highly sensitive real-time PCR.

We describe a highly sensitive real-time PCR to detect and measure the development of the liver-stages of malaria parasites in mice infected with sporozoites ranging in number from 25 to more than 164,000, using the same reaction conditions. Furthermore, this assay detects and measures parasite loads in the livers of mice exposed to the bite of a single malaria-infected Anopheles mosquito. This unique method should greatly facilitate studies aimed at evaluating very precisely the efficacy of anti-malarial experimental drug treatments and vaccination regimens in conditions of infection resembling those found in the field.

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.

IL-4-secreting CD4+ T cells are crucial to the development of CD8+ T-cell responses against malaria liver stages

CD4+ T cells are crucial to the development of CD8+ T cell responses against hepatocytes infected with malaria parasites. In the absence of CD4+ T cells, CD8+ T cells initiate a seemingly normal differentiation and proliferation during the first few days after immunization. However, this response fails to develop further and is reduced by more than 90%, compared to that observed in the presence of CD4+ T cells. We report here that interleukin-4 (IL-4) secreted by CD4+ T cells is essential to the full development of this CD8+ T cell response. This is the first demonstration that IL-4 is a mediator of CD4/CD8 cross-talk leading to the development of immunity against an infectious pathogen.

Multiple antigen peptide. A novel approach to increase detection sensitivity of synthetic peptides in solid-phase immunoassays.

We describe a novel approach to detect antibodies to synthetic peptide antigens in solid-phase radioimmunoassays, using a multiple antigen peptide (MAP) system. The MAPs consist of multiple copies of peptides that are synthesized as single units on a branching lysyl matrix using a solid-phase peptide synthesis method. The efficacy of the MAP approach in solid-phase immunoassays was compared with the conventional approach using a monomeric peptide of the immunodominant epitope of the circumsporozoite proteins of two species of malaria. Two monomeric peptides with 12 and 17 residues were found to bind poorly to plastic surfaces at a concentration up to 30 micrograms/ml, and showed no immunoreactivity to specific polyclonal or monoclonal antibodies, while the corresponding MAP-containing peptides showed excellent binding capacity and immunoreactivity at a concentration of 0.11 microgram/ml. The immunoreactivity of MAP-containing peptides was also superior to that of monomeric peptides conjugated to a protein carrier. The effects of various arrangements of lysyl branching of MAP on antigenicity were also studied, and the optimal number for lysyl branching of MAP was found to be octameric. Thus, the MAP, by enhancing the coating capacity and the avidity of synthetic peptides, provides increased sensitivity and reliability for the use of synthetic peptide to study antigen-antibody interactions on solid surfaces.

Demonstration of heat-shock protein 70 in the sporozoite stage of malaria parasites.

Three monoclonal antibodies generated by immunization of mice withPlasmodium berghei-infected red blood cells were found to react with the 75-kDa heat-shock protein (HSP70) present in liver stages and crythrocytic forms of the parasites. These antibodies were shown to react with a recombinant protein encoding the carboxyl terminal half of PfHSP70 (aa 365–681). Differently from earlier results, we clearly demonstrated that HSP70 was also expressed in the sporozoite stage, using these monoclonal antibodies in an immunofluorescence and Western immunoblot assay. These monoclonal antibodies react not only with sporozoites ofP. berghei, the parasites originally used for the immunization, but also with sporozoites of several other rodent and human plasmodial species. Passive transfer of these monoclonal antibodies into naive mice, simultaneously injected with sporozoites, failed to neutralize the infectivity ofP. berghei sporozoites and to inhibit the development of liver stages ofP. yoelii.

Protective CD8 T cells against Plasmodium liver stages: immunobiology of an 'unnatural' immune response.

Summary: Immunization with high doses of irradiated sporozoites delivered by the bites of infected mosquitoes has been shown to induce protective responses against malaria, mediated in part by CD8+ T cells. In contrast, natural transmission involving low exposure to live sporozoite antigen fails to elicit strong immunity. In this review, we examine how irradiated sporozoite immunization breaks the natural host–parasite interaction and induces protective CD8+ T cells. Upon biting, the malaria‐infected mosquitoes deposit parasites in the skin, many of which eventually exit to the bloodstream and infect hepatocytes. However, certain antigens, including the circumsporozoite (CS) protein, remain in the skin and are presented in the draining lymph node. These antigens prime specific CD8+ T cells, which migrate to the liver where they eliminate parasitized hepatocytes. We discuss the relevance of the different tissue compartments involved in the induction and effector phases of this response, as well as the cellular requirements for priming and memory development of CD8+ T cells, which include a complete dependence on dendritic cells and a near absolute need for CD4+ T‐cell help. Finally, we discuss the impact of the immunodominant CS protein on this protection and the implications of these findings for vaccine design.