Pre-erythrocytic Stage Malaria Research Articles

Evaluating the stability of host-reference gene expression and simultaneously quantifying parasite burden and host immune responses in murine malaria

The efficacy of pre-erythrocytic stage malaria antigens or vaccine platforms is routinely assessed in murine models challenged with Plasmodium sporozoites. Relative liver-stage parasite burden is quantified using reverse transcription quantitative PCR (RTqPCR), which relies on constitutively expressed endogenous control reference genes. However, the stability of host-reference gene expression for RTqPCR analysis following Plasmodium challenge and immunization has not been systematically evaluated. Herein, we evaluated the stability of expression of twelve common RTqPCR reference genes in a murine model of Plasmodium yoelii sporozoite challenge and DNA-adenovirus IV 'Prime-Target' immunization. Significant changes in expression for six of twelve reference genes were shown by one-way ANOVA, when comparing gene expression levels among challenge, immunized, and naïve mice groups. These changes were attributed to parasite challenge or immunization when comparing group means using post-hoc Bonferroni corrected multiple comparison testing. Succinate dehydrogenase (SDHA) and TATA-binding protein (TBP) were identified as stable host-reference genes suitable for relative RTqPCR data normalisation, using the RefFinder package. We defined a robust threshold of 'partial-protection’ with these genes and developed a strategy to simultaneously quantify matched host parasite burden and cytokine responses following immunisation or challenge. This is the first report systematically identifying reliable host reference genes for RTqPCR analysis following Plasmodium sporozoite challenge. A robust RTqPCR protocol incorporating reliable reference genes which enables simultaneous analysis of host whole-liver cytokine responses and parasite burden will significantly standardise and enhance results between international malaria vaccine efficacy studies.

A Critical Review on Human Malaria and Schistosomiasis Vaccines: Current State, Recent Advancements, and Developments.

Malaria and schistosomiasis are two major parasitic diseases that remain leading causes of morbidity and mortality worldwide. Co-infections of these two parasites are common in the tropics, where both diseases are endemic. The clinical consequences of schistosomiasis and malaria are determined by a variety of host, parasitic, and environmental variables. Chronic schistosomiasis causes malnutrition and cognitive impairments in children, while malaria can cause fatal acute infections. There are effective drugs available to treat malaria and schistosomiasis. However, the occurrence of allelic polymorphisms and the rapid selection of parasites with genetic mutations can confer reduced susceptibility and lead to the emergence of drug resistance. Moreover, the successful elimination and complete management of these parasites are difficult due to the lack of effective vaccines against Plasmodium and Schistosoma infections. Therefore, it is important to highlight all current vaccine candidates undergoing clinical trials, such as pre-erythrocytic and erythrocytic stage malaria, as well as a next-generation RTS,S-like vaccine, the R21/Matrix-M vaccine, that conferred 77% protection against clinical malaria in a Phase 2b trial. Moreover, this review also discusses the progress and development of schistosomiasis vaccines. Furthermore, significant information is provided through this review on the effectiveness and progress of schistosomiasis vaccines currently under clinical trials, such as Sh28GST, Sm-14, and Sm-p80. Overall, this review provides insights into recent progress in malarial and schistosomiasis vaccines and their developmental approaches.

Dissecting Plasmodium yoelii Pathobiology: Proteomic Approaches for Decoding Novel Translational and Post-Translational Modifications.

Malaria is a vector-borne disease. It is caused by Plasmodium parasites. Plasmodium yoelii is a rodent model parasite, primarily used for studying parasite development in liver cells and vectors. To better understand parasite biology, we carried out a high-throughput-based proteomic analysis of P. yoelii. From the same mass spectrometry (MS)/MS data set, we also captured several post-translational modified peptides by following a bioinformatics analysis without any prior enrichment. Further, we carried out a proteogenomic analysis, which resulted in improvements to some of the existing gene models along with the identification of several novel genes. Analysis of proteome and post-translational modifications (PTMs) together resulted in the identification of 3124 proteins. The identified PTMs were found to be enriched in mitochondrial metabolic pathways. Subsequent bioinformatics analysis provided an insight into proteins associated with metabolic regulatory mechanisms. Among these, the tricarboxylic acid (TCA) cycle and the isoprenoid synthesis pathway are found to be essential for parasite survival and drug resistance. The proteogenomic analysis discovered 43 novel protein-coding genes. The availability of an in-depth proteomic landscape of a malaria pathogen model will likely facilitate further molecular-level investigations on pre-erythrocytic stages of malaria.

Low immunogenicity of malaria pre-erythrocytic stages can be overcome by vaccination.

Immunogenicity is considered one important criterion for progression of candidate vaccines to further clinical evaluation. We tested this assumption in an infection and vaccination model for malaria pre‐erythrocytic stages. We engineered Plasmodium berghei parasites that harbour a well‐characterised epitope for stimulation of CD8+ T cells, either as an antigen in the sporozoite surface‐expressed circumsporozoite protein or the parasitophorous vacuole membrane associated protein upregulated in sporozoites 4 (UIS4) expressed in exo‐erythrocytic forms (EEFs). We show that the antigen origin results in profound differences in immunogenicity with a sporozoite antigen eliciting robust, superior antigen‐specific CD8+ T‐cell responses, whilst an EEF antigen evokes poor responses. Despite their contrasting immunogenic properties, both sporozoite and EEF antigens gain access to antigen presentation pathways in hepatocytes, as recognition and targeting by vaccine‐induced effector CD8+ T cells results in high levels of protection when targeting either antigen. Our study is the first demonstration that poorly immunogenic EEF antigens do not preclude their susceptibility to antigen‐specific CD8+ T‐cell killing, which has wide‐ranging implications on antigen prioritisation for next‐generation pre‐erythrocytic malaria vaccines.

Immune responses to malaria pre-erythrocytic stages: Implications for vaccine development.

Radiation-attenuated sporozoites induce sterilizing immunity and remain the ‘gold standard’ for malaria vaccine development. Despite practical challenges in translating these whole sporozoite vaccines to large-scale intervention programmes, they have provided an excellent platform to dissect the immune responses to malaria pre-erythrocytic (PE) stages, comprising both sporozoites and exoerythrocytic forms. Investigations in rodent models have provided insights that led to the clinical translation of various vaccine candidates—including RTS,S/AS01, the most advanced candidate currently in a trial implementation programme in three African countries. With advances in immunology, transcriptomics and proteomics, and application of lessons from past failures, an effective, long-lasting and wide-scale malaria PE vaccine remains feasible. This review underscores the progress in PE vaccine development, focusing on our understanding of host-parasite immunological crosstalk in the tissue environments of the skin and the liver. We highlight possible gaps in the current knowledge of PE immunity that can impact future malaria vaccine development efforts.

Importance of the Immunodominant CD8+T Cell Epitope of Plasmodium berghei Circumsporozoite Protein in Parasite- and Vaccine-Induced Protection

The circumsporozoite protein (CSP) builds up the surface coat of sporozoites and is the leading malaria pre-erythrocytic-stage vaccine candidate. CSP has been shown to induce robust CD8+ T cell responses that are capable of eliminating developing parasites in hepatocytes, resulting in protective immunity. In this study, we characterized the importance of the immunodominant CSP-derived epitope SYIPSAEKI of Plasmodium berghei in both sporozoite- and vaccine-induced protection in murine infection models. In BALB/c mice, where SYIPSAEKI is efficiently presented in the context of the major histocompatibility complex class I (MHC-I) molecule H-2-Kd, we established that epitope-specific CD8+ T cell responses contribute to parasite killing following sporozoite immunization. Yet, sterile protection was achieved in the absence of this epitope, substantiating the concept that other antigens can be sufficient for parasite-induced protective immunity. Furthermore, we demonstrated that SYIPSAEKI-specific CD8+ T cell responses elicited by viral-vectored CSP-expressing vaccines effectively targeted parasites in hepatocytes. The resulting sterile protection strictly relied on the expression of SYIPSAEKI. In C57BL/6 mice, which are unable to present the immunodominant epitope, CSP-based vaccines did not confer complete protection, despite the induction of high levels of CSP-specific antibodies. These findings underscore the significance of CSP in protection against malaria pre-erythrocytic stages and demonstrate that a significant proportion of the protection against the parasite is mediated by CD8+ T cells specific for the immunodominant CSP-derived epitope.

Innate immunity limits protective adaptive immune responses against pre-erythrocytic malaria infection.

Abstract Immunization with whole pre-erythrocytic stage malaria parasites that infect hepatocytes but are unable to cause blood stage infection constitutes one of the most promising strategies to protect against malaria infection. In rodent malaria models, whole parasite vaccines that exhibit substantial replication within hepatocytes engender superior, long-lasting strain- and stage-transcending immunity as compared to replication-deficient whole parasite vaccines. Liver stage infection with replication-competent Plasmodium parasites induces an innate immune response that is dependent on type I Interferon (IFN-1) signaling. However, it remains unknown how this IFN-1 response contributes to vaccine-engendered adaptive immunity. Using a replication-competent genetically attenuated parasite vaccine in an immunization regimen that confers partial protection in wildtype (WT) mice, we examined vaccine efficacy in mice lacking IRF3 or IFNAR. Surprisingly, IRF3−/− and IFNAR−/− mice showed superior protection against an infectious sporozoite challenge when compared to WT mice. Better protection correlated with a greater frequency of liver-resident memory CD8 T cells in immunized IFNAR−/− mice. Furthermore, IFNAR−/− mice exhibited lower levels of the T cell co-inhibitory receptors, PD-1 and LAG-3, a greater percentage of IFNγ-producing CD8 T cells, and eliminated liver stages more effectively. Taken together, we show that the Plasmodium engendered IFN-1 response impairs the generation of an optimal memory T cell response. To our knowledge, this study is the first description of a detrimental role for IFN-1 signaling on malaria vaccine efficacy and as such may necessitate a paradigm shift in rational malaria vaccine design.

Modeling the effect of boost timing in murine irradiated sporozoite prime-boost vaccines.

Vaccination with radiation-attenuated sporozoites has been shown to induce CD8+ T cell-mediated protection against pre-erythrocytic stages of malaria. Empirical evidence suggests that successive inoculations often improve the efficacy of this type of vaccines. An initial dose (prime) triggers a specific cellular response, and subsequent inoculations (boost) amplify this response to create a robust CD8+ T cell memory. In this work we propose a model to analyze the effect of T cell dynamics on the performance of prime-boost vaccines. This model suggests that boost doses and timings should be selected according to the T cell response elicited by priming. Specifically, boosting during late stages of clonal contraction would maximize T cell memory production for vaccines using lower doses of irradiated sporozoites. In contrast, single-dose inoculations would be indicated for higher vaccine doses. Experimental data have been obtained that support theoretical predictions of the model.

A Prime/Boost PfCS14K M /MVA-sPfCS M Vaccination Protocol Generates Robust CD8 + T Cell and Antibody Responses to Plasmodium falciparum Circumsporozoite Protein and Protects Mice against Malaria

Vaccines against the preerythrocytic stages of malaria are appealing because the parasite can be eliminated before disease onset and because they offer the unique possibility of targeting the parasite with both antibodies and T cells. Although the role of CD8+ T cells in preerythrocytic malaria stages is well documented, a highly effective T cell-inducing vaccine remains to be advanced. Here we report the development of a prime-boost immunization regimen with the Plasmodium falciparum circumsporozoite protein (PfCS) fused to the oligomer-forming vaccinia virus A27 protein and a modified vaccinia virus Ankara (MVA) vector expressing PfCS. This protocol induced polyfunctional CD8+ T cells with an effector memory phenotype and high PfCS antibody levels. These immune responses correlated with inhibition of liver-stage parasitemia in 80% and sterile protection in 40% of mice challenged with a transgenic P. berghei parasite line that expressed PfCS. Our findings underscore the potential of T and B cell immunization strategies for improving protective effectiveness against malaria.

Immunization of mice with live-attenuated late liver stage-arresting Plasmodium yoelii parasites generates protective antibody responses to preerythrocytic stages of malaria.

Understanding protective immunity to malaria is essential for the design of an effective vaccine to prevent the large number of infections and deaths caused by this parasitic disease. To date, whole-parasite immunization with attenuated parasites is the most effective method to confer sterile protection against malaria infection in clinical trials. Mouse model studies have highlighted the essential role that CD8(+) T cells play in protection against preerythrocytic stages of malaria; however, there is mounting evidence that antibodies are also important in these stages. Here, we show that experimental immunization of mice with Plasmodium yoelii fabb/f(-) (Pyfabb/f(-)), a genetically attenuated rodent malaria parasite that arrests late in the liver stage, induced functional antibodies that inhibited hepatocyte invasion in vitro and reduced liver-stage burden in vivo. These antibodies were sufficient to induce sterile protection from challenge by P. yoelii sporozoites in the absence of T cells in 50% of mice when sporozoites were administered by mosquito bite but not when they were administered by intravenous injection. Moreover, among mice challenged by mosquito bite, a higher proportion of BALB/c mice than C57BL/6 mice developed sterile protection (62.5% and 37.5%, respectively). Analysis of the antibody isotypes induced by immunization with Pyfabb/f(-) showed that, overall, BALB/c mice developed an IgG1-biased response, whereas C57BL/6 mice developed an IgG2b/c-biased response. Our data demonstrate for the first time that antibodies induced by experimental immunization of mice with a genetically attenuated rodent parasite play a protective role during the preerythrocytic stages of malaria. Furthermore, they highlight the importance of considering both the route of challenge and the genetic background of the mouse strains used when interpreting vaccine efficacy studies in animal models of malaria infection.

DNA from pre-erythrocytic stage malaria parasites is detectable by PCR in the faeces and blood of hosts

Following the bite of an infective mosquito, malaria parasites first invade the liver where they develop and replicate for a number of days before being released into the bloodstream where they invade red blood cells and cause disease. The biology of the liver stages of malaria parasites is relatively poorly understood due to the inaccessibility of the parasites to sampling during this phase of their life cycle. Here we report the detection in blood and faecal samples of malaria parasite DNA throughout their development in the livers of mice and before the parasites begin their growth in the blood circulation. It is shown that parasite DNA derived from pre-erythrocytic stage parasites reaches the faeces via the bile. We then show that different primate malaria species can be detected by PCR in blood and faecal samples from naturally infected captive macaque monkeys. These results demonstrate that pre-erythrocytic parasites can be detected and quantified in experimentally infected animals. Furthermore, these results have important implications for both molecular epidemiology and phylogenetics of malaria parasites. In the former case, individuals who are malaria parasite negative by microscopy, but PCR positive for parasite DNA in their blood, are considered to be “sub-microscopic” blood stage parasite carriers. We now propose that PCR positivity is not necessarily an indicator of the presence of blood stage parasites, as the DNA could derive from pre-erythrocytic parasites. Similarly, in the case of molecular phylogenetics based on DNA sequences alone, we argue that DNA amplified from blood or faeces does not necessarily come from a parasite species that infects the red blood cells of that particular host.

Intrasplenic immunization with infected hepatocytes: a mouse model for studying protective immunity against malaria pre‐erythrocytic stage

Malaria liver forms are the target of antibody or T-cell-mediated immune mechanisms induced by previous or subsequent developmental stages of the parasite. The potential for vaccine development of antigens expressed exclusively in the liver stages has not been fully explored partly because of the lack of an experimental animal model. Here we show that protective immunity against sporozoite-induced infection with Plasmodium yoelii and P. berghei can be obtained by intrasplenic injection of a small number of liver stages of the parasites. The serum of the protected animals did not contain antibodies against sporozoites, liver or blood stage malaria parasites. Protective immunity was abolished by depletion of either CD4+ or CD8+ T cells from the vaccinated mice before challenge.

Mixed Vector Immunization With Recombinant Adenovirus and MVA Can Improve Vaccine Efficacy While Decreasing Antivector Immunity

Substantial protection can be provided against the pre-erythrocytic stages of malaria by vaccination first with an adenoviral and then with an modified vaccinia virus Ankara (MVA) poxviral vector encoding the same ME.TRAP transgene. We investigated whether the two vaccine components adenovirus (Ad) and MVA could be coinjected as a mixture to enhance protection against malaria. A single-shot mixture at specific ratios of Ad and MVA (Ad+MVA) enhanced CD8+ T cell-dependant protection of mice against challenge with Plasmodium berghei. Moreover, the degree of protection could be enhanced after homologous boosting with the same Ad+MVA mixture to levels comparable with classic heterologous Ad prime-MVA boost regimes. The mixture increased transgene-specific responses while decreasing the CD8+ T cell antivector immunity compared to each vector used alone, particularly against the MVA backbone. Mixed vector immunization led to increased early circulating interferon-γ (IFN-γ) response levels and altered transcriptional microarray profiles. Furthermore, we found that sequential immunizations with the Ad+MVA mixture led to consistent boosting of the transgene-specific CD8+ response for up to three mixture immunizations, whereas each vector used alone elicited progressively lower responses. Our findings offer the possibility of simplifying the deployment of viral vectors as a single mixture product rather than in heterologous prime-boost regimens.

Dendritic cells and the malaria pre-erythrocytic stage

Malaria remains one of the main infectious diseases in intertropical regions. The malaria parasite has a complex life cycle in its mammalian host, switching between variable forms as it traverses through different tissues and anatomic locations, either intra- or intercellularly. During its journey, the parasite encounters and interacts with the host immune system, which functions to prevent infections and limit ensuing pathologies. One important component of the host immune system is the dendritic cells (DC) network. DC form a heterogeneous group of pathogen-sensing and antigen-presenting cells that play a crucial role in the initiation of adaptive immunity. Here, we review the known and unknown interactions between the malaria parasites and the DC system, starting from the inoculation of the parasite in the skin up to its exit from the liver, also known as the pre-erythrocytic stage of the infection, and discuss how deciphering these interactions may contribute to our understanding of the Plasmodium parasite biology as well as to the induction of immune protection via vaccination.

Early Transcriptional Responses of HepG2-A16 Liver Cells to Infection by Plasmodium falciparum Sporozoites

Invasion of hepatocytes by Plasmodium sporozoites deposited by Anopheles mosquitoes, and their subsequent transformation into infective merozoites is an obligatory step in the initiation of malaria. Interactions between the sporozoites and hepatocytes lead to a distinct, complex and coordinated cellular and systemic host response. Little is known about host liver cell response to sporozoite invasion, or whether it is primarily adaptive for the parasite, for the host, or for both. Our present study used gene expression profiling of human HepG2-A16 liver cells infected with Plasmodium falciparum sporozoites to understand the host early cellular events and factors influencing parasite infectivity and sporozoite development. Our results show that as early as 30 min following wild-type, non-irradiated sporozoite exposure, the expressions of at least 742 genes was selectively altered. These genes regulate diverse biological functions, such as immune processes, cell adhesion and communications, metabolism pathways, cell cycle regulation, and signal transduction. These functions reflect cellular events consistent with initial host cell defense responses, as well as alterations in host cells to sustain sporozoites growth and survival. Irradiated sporozoites gave very similar gene expression pattern changes, but direct comparative analysis between liver gene expression profiles caused by irradiated and non-irradiated sporozoites identified 29 genes, including glypican-3, that were specifically up-regulated only in irradiated sporozoites. Elucidating the role of this subset of genes may help identify the molecular basis for the irradiated sporozoites inability to develop intrahepatically, and their usefulness as an immunogen for developing protective immunity against pre-erythrocytic stage malaria.

Protective immunity to pre-erythrocytic stage malaria

The development of a vaccine against malaria is a major research priority given the burden of disease, death and economic loss inflicted upon the tropical world by this parasite. Despite decades of effort, however, a vaccine remains elusive. The best candidate is a subunit vaccine termed RTS,S but this provides only partial protection against clinical disease. This review examines what is known about protective immunity against pre-erythrocytic stage malaria by considering the humoral and T cell-mediated immune responses that are induced by attenuated sporozoites and by the RTS,S vaccine. On the basis of these observations a set of research priorities are defined that are crucial for the development of a vaccine capable of inducing long-lasting and high-grade protection against malaria.

Identification of two new protective pre-erythrocytic malaria vaccine antigen candidates

BackgroundDespite years of effort, a licensed malaria vaccine is not yet available. One of the obstacles facing the development of a malaria vaccine is the extensive heterogeneity of many of the current malaria vaccine antigens. To counteract this antigenic diversity, an effective malaria vaccine may need to elicit an immune response against multiple malaria antigens, thereby limiting the negative impact of variability in any one antigen. Since most of the malaria vaccine antigens that have been evaluated in people have not elicited a protective immune response, there is a need to identify additional protective antigens. In this study, the efficacy of three pre-erythrocytic stage malaria antigens was evaluated in a Plasmodium yoelii/mouse protection model.MethodsMice were immunized with plasmid DNA and vaccinia virus vectors that expressed one, two or all three P. yoelii vaccine antigens. The immunized mice were challenged with 300 P. yoelii sporozoites and evaluated for subsequent infection.ResultsVaccines that expressed any one of the three antigens did not protect a high percentage of mice against a P. yoelii challenge. However, vaccines that expressed all three antigens protected a higher percentage of mice than a vaccine that expressed PyCSP, the most efficacious malaria vaccine antigen. Dissection of the multi-antigen vaccine indicated that protection was primarily associated with two of the three P. yoelii antigens. The protection elicited by a vaccine expressing these two antigens exceeded the sum of the protection elicited by the single antigen vaccines, suggesting a potential synergistic interaction.ConclusionsThis work identifies two promising malaria vaccine antigen candidates and suggests that a multi-antigen vaccine may be more efficacious than a single antigen vaccine.

Inhibitory Effect of TNF-α on Malaria Pre-Erythrocytic Stage Development: Influence of Host Hepatocyte/Parasite Combinations

BackgroundThe liver stages of malaria parasites are inhibited by cytokines such as interferon-γ or Interleukin (IL)-6. Binding of these cytokines to their receptors at the surface of the infected hepatocytes leads to the production of nitric oxide (NO) and radical oxygen intermediates (ROI), which kill hepatic parasites. However, conflicting results were obtained with TNF-α possibly because of differences in the models used. We have reassessed the role of TNF-α in the different cellular systems used to study the Plasmodium pre-erythrocytic stages.Methods and FindingsHuman or mouse TNF-α were tested against human and rodent malaria parasites grown in vitro in human or rodent primary hepatocytes, or in hepatoma cell lines. Our data demonstrated that TNF-α treatment prevents the development of malaria pre-erythrocytic stages. This inhibitory effect however varies with the infecting parasite species and with the nature and origin of the cytokine and hepatocytes. Inhibition was only observed for all parasite species tested when hepatocytes were pre-incubated 24 or 48 hrs before infection and activity was directed only against early hepatic parasite. We further showed that TNF-α inhibition was mediated by a soluble factor present in the supernatant of TNF-α stimulated hepatocytes but it was not related to NO or ROI. Treatment TNF-α prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified.ConclusionsTreatment TNF-α prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified. However, the nature of the cytokine-host cell-parasite combination must be carefully considered for extrapolation to the human infection.

Interferon-γ, a valuable surrogate marker of Plasmodium falciparum pre-erythrocytic stages protective immunity

Immunity against the pre-erythrocytic stages of malaria is the most promising, as it is strong and fully sterilizing. Yet, the underlying immune effectors against the human Plasmodium falciparum pre-erythrocytic stages remain surprisingly poorly known and have been little explored, which in turn prevents any rational vaccine progress. Evidence that has been gathered in vitro and in vivo, in higher primates and in humans, is reviewed here, emphasizing the significant role of IFN-γ, either as a critical immune mediator or at least as a valuable surrogate marker of protection. One may hope that these results will trigger investigations in volunteers immunized either by optimally irradiated or over-irradiated sporozoites, to quickly delineate better surrogates of protection, which are essential for the development of a successful malaria vaccine.

The intradermal route for inoculation of sporozoites of rodent malaria parasites for immunological studies

Rodent malaria parasites are commonly used for investigations into the immunology of pre-erythrocytic stage malaria infection, as sporozoites can be easily produced in the laboratory. In the majority of past immunological studies using this system, sporozoites are inoculated into mice via the intravenous (IV) route. In natural situations, however, sporozoites are deposited into the skin by the bite of Anopheline mosquitoes, and it is likely that the immunological response to such natural intradermal (ID) inoculation will be different to that achieved through the IV route. Although infected mosquito bites are sometimes used during experimental induction of immunity in mice, this method is problematic because of the low numbers of sporozoites introduced to the skin and the large variation in sporozoite inoculation between individual mosquitoes. Here, we show that ID inoculation of dissected mosquito salivary gland sporozoites of Plasmodium yoelii allows the accurate introduction of known numbers of sporozoites into the skin and that these parasites successfully invade the liver. Furthermore, immunization of mice using ID inoculations of live sporozoites contemporaneously with mefloquine treatment induces an immune response that is protective against the development of liver stage parasites, and this protection does not differ significantly from that achieved with IV immunizations performed in the same manner.