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.     

Combining liver- and blood-stage malaria viral-vectored vaccines: investigating mechanisms of CD8+ T cell interference

Replication-deficient adenovirus and modified vaccinia virus Ankara (MVA) vectors expressing single pre-erythrocytic or blood-stage Plasmodium falciparum Ags have entered clinical testing using a heterologous prime-boost immunization approach. In this study, we investigated the utility of the same immunization regimen when combining viral vectored vaccines expressing the 42-kDa C terminus of the blood-stage Ag merozoite surface protein 1 and the pre-erythrocytic Ag circumsporozoite protein in the Plasmodium yoelii mouse model. We find that vaccine coadministration leads to maintained Ab responses and efficacy against blood-stage infection, but reduced secondary CD8(+) T cell responses against both Ags and efficacy against liver-stage infection. CD8(+) T cell interference can be minimized by coadministering the MVA vaccines at separate sites, resulting in enhanced liver-stage efficacy in mice immunized against both Ags compared with just one. CD8(+) T cell interference (following MVA coadministration as a mixture) may be caused partly by a lack of physiologic space for high-magnitude responses against multiple Ags, but is not caused by competition for presentation of Ag on MHC class I molecules, nor is it due to restricted T cell access to APCs presenting both Ags. Instead, enhanced killing of peptide-pulsed cells is observed in mice possessing pre-existing T cells against two Ags compared with just one, suggesting that priming against multiple Ags may in part reduce the potency of multiantigen MVA vectors to stimulate secondary CD8(+) T cell responses. These data have important implications for the development of a multistage or multicomponent viral vectored malaria vaccine for use in humans.     

High quality long-term CD4+ and CD8+ effector memory populations stimulated by DNA-LACK/MVA-LACK regimen in Leishmania major BALB/c model of infection

Heterologous vaccination based on priming with a plasmid DNA vector and boosting with an attenuated vaccinia virus MVA recombinant, with both vectors expressing the Leishmania infantum LACK antigen (DNA-LACK and MVA-LACK), has shown efficacy conferring protection in murine and canine models against cutaneus and visceral leishmaniasis, but the immune parameters of protection remain ill defined. Here we performed by flow cytometry an in depth analysis of the T cell populations induced in BALB/c mice during the vaccination protocol DNA-LACK/MVA-LACK, as well as after challenge with L. major parasites. In the adaptive response, there is a polyfunctional CD4(+) and CD8(+) T cell activation against LACK antigen. At the memory phase the heterologous vaccination induces high quality LACK-specific long-term CD4(+) and CD8(+) effector memory cells. After parasite challenge, there is a moderate boosting of LACK-specific CD4(+) and CD8(+) T cells. Anti-vector responses were largely CD8(+)-mediated. The immune parameters induced against LACK and triggered by the combined vaccination DNA/MVA protocol, like polyfunctionality of CD4(+) and CD8(+) T cells with an effector phenotype, could be relevant in protection against leishmaniasis.     

Enhancing blood-stage malaria subunit vaccine immunogenicity in rhesus macaques by combining adenovirus, poxvirus, and protein-in-adjuvant vaccines

Protein-in-adjuvant formulations and viral-vectored vaccines encoding blood-stage malaria Ags have shown efficacy in rodent malaria models and in vitro assays against Plasmodium falciparum. Abs and CD4(+) T cell responses are associated with protective efficacy against blood-stage malaria, whereas CD8(+) T cells against some classical blood-stage Ags can also have a protective effect against liver-stage parasites. No subunit vaccine strategy alone has generated demonstrable high-level efficacy against blood-stage infection in clinical trials. The induction of high-level Ab responses, as well as potent T and B cell effector and memory populations, is likely to be essential to achieve immediate and sustained protective efficacy in humans. This study describes in detail the immunogenicity of vaccines against P. falciparum apical membrane Ag 1 in rhesus macaques (Macaca mulatta), including the chimpanzee adenovirus 63 (AdCh63), the poxvirus modified vaccinia virus Ankara (MVA), and protein vaccines formulated in Alhydrogel or CoVaccine HT adjuvants. AdCh63-MVA heterologous prime-boost immunization induces strong and long-lasting multifunctional CD8(+) and CD4(+) T cell responses that exhibit a central memory-like phenotype. Three-shot (AdCh63-MVA-protein) or two-shot (AdCh63-protein) regimens induce memory B cells and high-titer functional IgG responses that inhibit the growth of two divergent strains of P. falciparum in vitro. Prior immunization with adenoviral vectors of alternative human or simian serotype does not affect the immunogenicity of the AdCh63 apical membrane Ag 1 vaccine. These data encourage the further clinical development and coadministration of protein and viral vector vaccine platforms in an attempt to induce broad cellular and humoral immune responses against blood-stage malaria Ags in humans.     

Protracted protection to Plasmodium berghei malaria is linked to functionally and phenotypically heterogeneous liver memory CD8+ T cells

We previously demonstrated that protection induced by radiation-attenuated (gamma) Plasmodium berghei sporozoites is linked to MHC class I-restricted CD8(+) T cells specific for exoerythrocytic-stage Ags, and that activated intrahepatic memory CD8(+) T cells are associated with protracted protection. In this study, we further investigated intrahepatic memory CD8(+) T cells to elucidate mechanisms required for their maintenance. Using phenotypic markers indicative of activation (CD44, CD45RB), migration (CD62L), and IFN-gamma production, we identified two subsets of intrahepatic memory CD8(+) T cells: the CD44(high)CD45RB(low)CD62L(low)CD122(low) phenotype, representing the dominant effector memory set, and the CD44(high)CD45RB(high)CD62L(low/high)CD122(high) phenotype, representing the central memory set. Only the effector memory CD8(+) T cells responded swiftly to sporozoite challenge by producing sustained IFN-gamma; the central memory T cells responded with delay, and the IFN-gamma reactivity was short-lived. In addition, the subsets of liver memory CD8(+) T cells segregated according to the expression of CD122 (IL-15R) in that only the central memory CD8(+) T cells were CD122(high), whereas the effector memory CD8(+) T cells were CD122(low). Moreover, the effector memory CD8(+) T cells declined as protection waned in mice treated with primaquine, a drug that interferes with the formation of liver-stage Ags. We propose that protracted protection induced by P. berghei radiation-attenuated sporozoites depends in part on a network of interactive liver memory CD8(+) T cell subsets, each representing a different phase of activation or differentiation, and the balance of which is profoundly affected by the repository of liver-stage Ag and IL-15.     

Vaccine efficacy against malaria by the combination of porcine parvovirus-like particles and vaccinia virus vectors expressing CS of Plasmodium

With the aim to develop an efficient and cost-effective approach to control malaria, we have generated porcine parvovirus-like particles (PPV-VLPs) carrying the CD8(+) T cell epitope (SYVPSAEQI) of the circumsporozoite (CS) protein from Plasmodium yoelii fused to the PPV VP2 capsid protein (PPV-PYCS), and tested in prime/boost protocols with poxvirus vectors for efficacy in a rodent malaria model. As a proof-of concept, we have characterized the anti-CS CD8(+) T cell response elicited by these hybrid PPV-VLPs in BALB/c mice after immunizations with the protein PPV-PYCS administered alone or in combination with recombinant vaccinia virus (VACV) vectors from the Western Reserve (WR) and modified virus Ankara (MVA) strains expressing the entire P. yoelii CS protein. The results of different immunization protocols showed that the combination of PPV-PYCS prime/poxvirus boost was highly immunogenic, inducing specific CD8+ T cell responses to CS resulting in 95% reduction in liver stage parasites two days following sporozoite challenge. In contrast, neither the administration of PPV-PYCS alone nor the immunization with the vectors given in the order poxvirus/VLPs was as effective. The immune profile induced by VLPs/MVA boost was associated with polyfunctional and effector memory CD8+ T cell responses. These findings highlight the use of recombinant parvovirus PPV-PYCS particles as priming agents and poxvirus vectors, like MVA, as booster to enhance specific CD8+ T cell responses to Plasmodium antigens and to control infection. These observations are relevant in the design of T cell-inducing vaccines against malaria.     

Protective CD8+ T cell responses against the pre-erythrocytic stages of malaria parasites: an overview

CD8+ T cells have been implicated as critical effector cells in protection against the pre-erythrocytic stage of malaria in mice and humans following irradiated sporozoite immunization. Immunization experiments in animal models by several investigators have suggested different strategies for vaccination against malaria and many of the targets from liver stage malaria antigens have been shown to be immunogenic and to protect mice from the sporozoite challenge. Several prime/boost protocols with replicating vectors, such as vaccinia/influenza, with non-replicating vectors, such as recombinant particles derived from yeast transposon (Ty-particles) and modified vaccinia virus Ankara, and DNA, significantly enhanced CD8+ T cell immunogenicity and also the protective efficacy against the circumsporosoite protein of Plasmodium berghei and P. yeti. Based on these experimental results the development of a CD8+ T cell inducing vaccine has moved forward from epitope identification to planning stages of safety and immunogenicity trials of candidate vaccines.     

Splenic dendritic cell subsets prime and boost CD8 T cells and are involved in the generation of effector CD8 T cells

The ability of the dendritic cell (DC) subsets, CD8alpha+ and CD8alpha- DCs, to initiate a CD8 T cell response or to activate memory CD8 T cells and generate effector CD8 T cells has been controversial. In this study, we analyse the capacity of splenic DC subsets to induce CD8 T cell responses to a CD8 T cell epitope (pb9) of a malaria antigen. The administration of peptide-pulsed CD8alpha- or CD8alpha+ DCs primes and boosts a primed CD8 T cell response against the malaria epitope. In vitro, depletion of CD11c(+) DCs from mouse splenocytes, immunised with recombinant vaccinia virus Ankara (MVA) expressing pb9 epitope, significantly reduced the generation of pb9-specific IFNgamma producing effector CD8 T cells, indicating that splenic DCs are involved in the development of pb9-specific IFNgamma producing effector cells. Taken together, this result shows that both DC subsets have the ability to prime and boost CD8 T cell responses and are involved in the activation of memory CD8 T cells.     

T cells as mediators of protective immunity against liver stages of Plasmodium

T cells from different subsets play a major role in protective immunity against pre-erythrocytic stages of malaria parasites. Exposure of humans and animals to malaria sporozoites induces (alphabeta CD8(+) and CD4(+) T cells specific for antigens expressed in pre-erythrocytic stages of Plasmodium. These T cells inhibit parasite development in the liver, and immunization with subunit vaccines expressing the respective antigenic moieties confers protection against sporozoite challenge. gammadelta and natural killer T cells can also play a role in protective immunity. Recent studies with mice transgenic for the alphabeta T-cell receptor have revealed the existence of complex mechanisms regulating the induction and development of these responses.     

Differential immunogenicity of various heterologous prime-boost vaccine regimens using DNA and viral vectors in healthy volunteers

Heterologous prime-boost vaccination has been shown to be an efficient way of inducing T cell responses in animals and in humans. We have used three vaccine vectors, naked DNA, modified vaccinia virus Ankara (MVA), and attenuated fowlpox strain, FP9, for prime-boost vaccination approaches against Plasmodium falciparum malaria in humans. In this study, we characterize, using two types of ELISPOT assays and FACS analysis, cell-mediated immune responses induced by different prime-boost combinations where all vectors encode a multiepitope string fused to the pre-erythrocytic Ag thrombospondin-related adhesion protein. We show that these different vectors need to be used in a specific order for an optimal ex vivo IFN-gamma response. From the different combinations, DNA priming followed by MVA boosting and FP9 priming followed by MVA boosting were most immunogenic and in both cases the IFN-gamma response was of broad specificity and cross-reactive against two P. falciparum strains (3D7 and T9/96). Immunization with all three vectors showed no improvement over optimal two vector regimes. Strong ex vivo IFN-gamma responses peaked 1 wk after the booster dose, but cultured ELISPOT assays revealed longer-lasting T cell memory responses for at least 6 mo. In the DNA-primed vaccinees the IFN-gamma response was mainly due to CD4(+) T cells, whereas in the FP9-primed vaccinees it was mainly due to CD4-dependent CD8(+) T cells. This difference may be of importance for the protective efficacy of these vaccination approaches against various diseases.     

Depletion of CD4+ T cells during immunization with nonviable Listeria monocytogenes causes enhanced CD8+ T cell-mediated protection against listeriosis

Immunization of mice with nonviable Listeria monocytogenes generates an insufficient CD8(+) T cell response and consequently only limited protection against subsequent L. monocytogenes infection. We have recently demonstrated that depletion of regulatory CD4(+) T cells during immunization significantly enhances CD8(+) T cell responses. In the present study, we determined the impact of CD4(+) T cell depletion on the CD8(+) T cell response against heat-killed LISTERIA: Treatment of mice with anti-CD4 mAb during boost immunization with heat-killed Listeria significantly increased numbers of Listeria-specific CD8(+) T cells and improved protection against subsequent infection with L. monocytogenes. During challenge infection, numbers of Listeria-specific CD8(+) T cells were enhanced, and these cells expressed effector functions in terms of IFN-gamma production. In summary, we demonstrate that combining nonviable L. monocytogenes vaccination and CD4(+) T cell depletion improves generation of long-lasting and functional Listeria-specific CD8(+) memory T cells.     

KLRG1+NKG2A+ CD8 T cells mediate protection and participate in memory responses during γ-herpesvirus infection

Functional CD8 T cell effector and memory responses are generated and maintained during murine γ-herpesvirus 68 (γHV68) persistent infection despite continuous presentation of viral lytic Ags. However, the identity of the CD8 T cell subpopulations that mediate effective recall responses and that can participate in the generation of protective memory to a γ-herpesvirus infection remains unknown. During γHV68 persistence, ～75% of γHV68-specific CD8 T cells coexpress the NK receptors killer cell lectin-like receptor G1 (KLRG1) and NKG2A. In this study, we take advantage of this unique phenotype to analyze the capacity of CD8 T cells expressing or not expressing KLRG1 and NKG2A to mediate effector and memory responses. Our results show that γHV68-specific KLRG1(+)NKG2A(+) CD8 T cells have an effector memory phenotype as well as characteristics of polyfunctional effector cells such us IFN-γ and TNF-α production, killing capacity, and are more efficient at protecting against a γHV68 challenge than their NKG2A(-)KLRG1(-) counterparts. Nevertheless, γHV68-specific NKG2A(+)KLRG1(+) CD8 T cells express IL-7 and IL-15 receptors, can survive long-term without cognate Ag, and subsequently mount a protective response during antigenic recall. These results highlight the plasticity of the immune system to generate protective effector and proliferative memory responses during virus persistence from a pool of KLRG1(+)NKG2A(+) effector memory CD8 T cells.     

Enhancement of CD8+ T cell immunity in the lung by CpG oligodeoxynucleotides increases protective efficacy of a modified vaccinia Ankara vaccine against lethal poxvirus infection even in a CD4-deficient host

Immunostimulatory CpG oligodeoxynucleotides (ODN) have proven effective as adjuvants for protein-based vaccines, but their impact on immune responses induced by live viral vectors is not known. We found that addition of CpG ODN to modified vaccinia Ankara (MVA) markedly improved the induction of longer-lasting adaptive protective immunity in BALB/c mice against intranasal pathogenic vaccinia virus (Western Reserve; WR). Protection was mediated primarily by CD8(+) T cells in the lung, as determined by CD8-depletion studies, protection in B cell-deficient mice, and greater protection correlating with CD8(+) IFN-gamma-producing cells in the lung but not with those in the spleen. Intranasal immunization was more effective at inducing CD8(+) T cell immunity in the lung, and protection, than i.m. immunization. Addition of CpG ODN increased the CD8(+) response but not the Ab response. Depletion of CD4 T cells before vaccination with MVA significantly diminished protection against pathogenic WR virus. However, CpG ODN delivered with MVA was able to substitute for CD4 help and protected CD4-depleted mice against WR vaccinia challenge. This study demonstrates for the first time a protective adjuvant effect of CpG ODN for a live viral vector vaccine that may overcome CD4 deficiency in the induction of protective CD8(+) T cell-mediated immunity.     

Differential requirements for OX40 signals on generation of effector and central memory CD4+ T cells

Memory T cells can be divided into effector memory (T(EM)) and central memory (T(CM)) subsets based on their effector function and homing characteristics. Although previous studies have demonstrated that TCR and cytokine signals mediate the generation of the two memory subsets of CD8(+) T cells, the mechanisms for generation of the CD4(+) T(EM) and T(CM) cell subsets are unknown. We found that OX40-deficient mice showed a marked reduction in the number of CD4(+) T(EM) cells, whereas the number of CD4(+) T(CM) cells was normal. Adoptive transfer experiments using Ag-specific CD4(+) T cells revealed that OX40 signals during the priming phase were indispensable for the optimal generation of the CD4(+) T(EM), but not the CD4(+) T(CM) population. In a different transfer experiment with in vitro established CD4(+)CD44(high)CD62L(low) (T(EM) precursor) and CD4(+)CD44(high)CD62L(high) (T(CM) precursor) subpopulations, OX40-KO T(EM) precursor cells could not survive in the recipient mice, whereas wild-type T(EM) precursor cells differentiated into both T(EM) and T(CM) cells. In contrast, T(CM) precursor cells mainly produced T(CM) cells regardless of OX40 signals, implying the dispensability of OX40 for generation of T(CM) cells. Nevertheless, survival of OX40-KO T(EM) cells was partially rescued in lymphopenic mice. During in vitro recall responses, the OX40-KO T(EM) cells that were generated in lymphopenic recipient mice showed impaired cytokine production, suggesting an essential role for OX40 not only on generation but also on effector function of CD4(+) T(EM) cells. Collectively, the present results indicate differential requirements for OX40 signals on generation of CD4(+) T(EM) and T(CM) cells.     

Preferential expansion of human virus-specific multifunctional central memory T cells by partial targeting of the IL-2 receptor signaling pathway: the key role of CD4+ T cells

Effector memory T cells are effective in controlling acute infections, but central memory T cells play a key role in long-lasting protection against viruses and tumors. In vivo/in vitro challenge by Ag commonly supports the generation of effector memory T cells with limited longevity. To our knowledge, this study demonstrates for the first time in the human system and under rechallenge conditions that targeting IL-2R by partial mammalian target of rapamycin inhibition or blocking IL-2Rα enriches human CD4(+)/CD8(+) central memory T cells within the virus-specific T cell product associated with enhanced functionality (i.e., multicytokine secretors, including IL-2; enhanced CD137 and CD107a expression on CD8(+) and CD4(+) T cells, respectively; and killing infected target cells). Remarkably, the effects on CD8(+) T cells are mainly mediated via the enhancement of CD4(+) T cell function. The data reveal new insights into the role of CD4(+) T cell support for the quality of CD8(+) T cell memory, even under rechallenge conditions. Moreover, our method offers a new approach to improve the long-lasting efficacy of adoptive T cell therapy in patients.     

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.     

Retinoic acid as a vaccine adjuvant enhances CD8+ T cell response and mucosal protection from viral challenge

Vaccine-induced memory T cells localized at mucosal sites can provide rapid protection from viral infection. All-trans-retinoic acid (ATRA) has been shown to act physiologically to induce the expression of gut-homing receptors on lymphocytes. We tested whether the administration of exogenous ATRA during a systemic vaccination of mice could enhance the generation of mucosal CD8(+) T cell immunity, which might represent a strategy for establishing better protection from viral infection via mucosal routes. ATRA induced the expression of CCR9 and α4β7 on both mouse and human CD8(+) T cells activated in vitro. The administration of ATRA to mice during in vivo priming with a replication-defective recombinant adenovirus vector expressing the lymphocytic choriomeningitis virus glycoprotein (LCMVgp) (Ad5gp) increased numbers of both effector and memory T cells in intestinal mucosal tissues and showed higher frequencies of systemic central memory-like T cells that exhibited enhanced proliferation during boosting immunization with recombinant modified vaccinia virus Ankara expressing LCMVgp (MVAgp). Mice that received ATRA during Ad5gp vaccination were more resistant to intravaginal challenge by recombinant vaccinia virus expressing LCMVgp (VVgp), reflecting in part stronger T cell recall responses in situ. Thus, ATRA appears to be useful as an adjuvant during vaccination to increase memory T cell responses and protection from viral infection at mucosal sites and may facilitate the development of more effective vaccines against mucosally transmitted pathogens such as HIV.     

Effector memory Th1 CD4 T cells are maintained in a mouse model of chronic malaria

Protection against malaria often decays in the absence of infection, suggesting that protective immunological memory depends on stimulation. Here we have used CD4(+) T cells from a transgenic mouse carrying a T cell receptor specific for a malaria protein, Merozoite Surface Protein-1, to investigate memory in a Plasmodium chabaudi infection. CD4(+) memory T cells (CD44(hi)IL-7Rα(+)) developed during the chronic infection, and were readily distinguishable from effector (CD62L(lo)IL-7Rα(-)) cells in acute infection. On the basis of cell surface phenotype, we classified memory CD4(+) T cells into three subsets: central memory, and early and late effector memory cells, and found that early effector memory cells (CD62L(lo)CD27(+)) dominated the chronic infection. We demonstrate a linear pathway of differentiation from central memory to early and then late effector memory cells. In adoptive transfer, CD44(hi) memory cells from chronically infected mice were more effective at delaying and reducing parasitemia and pathology than memory cells from drug-treated mice without chronic infection, and contained a greater proportion of effector cells producing IFN-γ and TNFα, which may have contributed to the enhanced protection. These findings may explain the observation that in humans with chronic malaria, activated effector memory cells are best maintained in conditions of repeated exposure.     

Immune response to pre-erythrocytic stages of malaria parasites

Immunization with radiation-attenuated Plasmodium spp. sporozoites induces sterile protective immunity against parasite challenge. This immunity is targeted primarily against the intrahepatic parasite and appears to be sustained long term even in the absence of sporozoite exposure. It is mediated by multifactorial mechanisms, including T cells directed against parasite antigens expressed in the liver stage of the parasite life cycle and antibodies directed against sporozoite surface proteins. In rodent models, CD8+ T cells have been implicated as the principal effector cells, and IFN-gamma as a critical effector molecule. IL-4 secreting CD4+ T cells are required for induction of the CD8+ T cell responses, and Th1 CD4+ T cells provide help for optimal CD8+ T cell effector activity. Components of the innate immune system, including gamma-delta T cells, natural killer cells and natural killer T cells, also play a role. The precise nature of pre-erythrocytic stage immunity in humans, including the contribution of these immune responses to the age-dependent immunity naturally acquired by residents of malaria endemic areas, is still poorly defined. The importance of immune effector targets at the pre-erythrocytic stage of the parasite life cycle is highlighted by the fact that infection-blocking immunity in humans rarely, if ever, occurs under natural conditions. Herein, we review our current understanding of the molecular and cellular aspects of pre-erythrocytic stage immunity.