A highly efficacious malaria vaccine that prevents disease and breaks the cycle of infection remains an aspirational goal of medicine. Whole parasite vaccines based on the sporozoite forms of the parasite that target the clinically silent pre-erythrocytic stages of infection have emerged as one of the leading candidates. In animal models of malaria, these vaccines elicit potent neutralizing Ab responses against the sporozoite stage and cytotoxic T cells that eliminate parasite-infected hepatocytes. Among whole-sporozoite vaccines, immunization with live, replication-competent whole parasites engenders superior immunity and protection when compared with live replication-deficient sporozoites. As such, the genetic design of replication-competent vaccine strains holds the promise for a potent, broadly protective malaria vaccine. In this report, we will review the advances in whole-sporozoite vaccine development with a particular focus on genetically attenuated parasites both as malaria vaccine candidates and also as valuable tools to interrogate protective immunity against Plasmodium infection.

An estimated 216 million new clinical cases of malaria were reported in 2016, 445,000 of which were fatal (World Health Organization [WHO] 2017). The causative pathogens for malaria are complex obligate intracellular Plasmodium parasites that are transmitted to humans by female Anopheles mosquito vectors. There are five different species of Plasmodium parasites that cause malaria in humans: Plasmodium falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi. These species have both shared and distinct geographical distributions (WHO 2017), with P. falciparum having the highest prevalence in sub-Saharan Africa, whereas P. vivax is the predominant parasite species outside of Africa (WHO 2017). Over the last decade, the increased implementation of existing malaria control tools and treatment interventions has significantly reduced malaria-associated morbidity and mortality. However, these gains have stagnated in regions of the world with the highest transmission rates, such as sub-Saharan Africa. Additionally, the continuous emergence of drug resistance to frontline antimalarial drugs renders drug development efforts a never-ending arms race. Therefore, novel antimalarial interventions that constitute more sustainable solutions, such as a protective antimalarial vaccine, are needed to prevent disease and break the cycle of transmission.

Transmission of parasites from an infected mosquito vector to the mammalian host begins with the deposition of infectious sporozoite forms into the skin (Fig. 1A). Sporozoites gain access to the bloodstream to reach and enter the liver. (Fig. 1B) (14). In the liver, each sporozoite then infects a single hepatocyte, within which it transforms into a liver stage parasite, undergoes growth, and differentiates into tens of thousands of exoerythrocytic merozoites (Fig. 1Ci). Merozoites are released from the liver into the bloodstream 7–10 d after initial transmission to initiate a continuous cycle of RBC infection (Fig. 1E), replication, and release, allowing parasite numbers to reach billions within days to weeks. The sporozoite and liver stages of infection, together termed the pre-erythrocytic stages, are asymptomatic, whereas the blood stage (BS) infection is responsible for all malaria-associated morbidity and mortality. Closing the cycle, uptake of parasite sexual forms in a blood meal leads to infection of the mosquito, and accumulation of infectious sporozoites in the mosquito salivary glands ensures transmission to new human hosts upon the next blood meal (5, 6).

FIGURE 1.

Plasmodium infection and the host immune responses that control infection after whole-sporozoite immunization. (A) Infection in humans begins when an infected mosquito bite deposits sporozoites in the skin. Immunization with whole-sporozoite vaccines elicits Abs that immobilize sporozoites in the skin and prevent exit of sporozoites into the nearest blood vessel. (B) The sporozoites that successfully enter circulation transmigrate out of the liver sinusoidal endothelium and traverse multiple cell types until they can successfully invade hepatocytes. Abs can block extravasation and infection of hepatocytes. (Ci) Within each infected hepatocyte, the sporozoite transforms into a trophozoite stage, followed by cell growth and DNA replication during schizogony to ultimately generate tens of thousands of exoerythrocytic merozoites. Cytotoxic T cells and cytokine signaling can eliminate parasite-infected hepatocytes or eliminate the parasite within the hepatocyte, respectively. (ii) RAS and EARD GAPs arrest at the trophozoite/early schizont stage within hepatocytes. Because of this early developmental arrest, immunizations with RAS and EARD GAPs expose the immune system to a repertoire of Ags that is limited to sporozoites and early liver stages. (iii) LARC GAPs, in comparison, undergo nearly full schizogony and arrest late in development. LARC GAPs have large parasite biomass and express a broad repertoire of Ags encompassing sporozoite, early liver–stage, and late live–stage Ags, some of which are shared with BSs. During CPS vaccination (i), parasites undergo full schizogony within hepatocytes. Exoerythrocytic merozoites are released into circulation, and they invade RBCs, but chloroquine kills the parasite within RBCs. Immunizations with CPS primes the host with a broad repertoire of sporozoite, liver-stage, and BS Ags. (D) Immunizations with whole-sporozoite vaccines triggers potent cellular adaptive immunity to kill infected hepatocytes. Additionally, they drive a potent recruitment of myeloid cells and innate lymphoid cells into the liver. (E) Upon egress from the liver, merozoites initiate a 48-h cycle of infection, asexual replication, and destruction of RBCs. Some asexual parasites commit to gametocytogenesis and generate male and female gametocytes, which are then taken up by an Anopheles mosquito during a blood meal and the cycle continues. Immunization with RC whole-sporozoite vaccines, such as CPS or LARC GAP, induces both humoral and cellular immune responses against pre-erythrocytic– and erythrocytic-stage parasites.

FIGURE 1.

Plasmodium infection and the host immune responses that control infection after whole-sporozoite immunization. (A) Infection in humans begins when an infected mosquito bite deposits sporozoites in the skin. Immunization with whole-sporozoite vaccines elicits Abs that immobilize sporozoites in the skin and prevent exit of sporozoites into the nearest blood vessel. (B) The sporozoites that successfully enter circulation transmigrate out of the liver sinusoidal endothelium and traverse multiple cell types until they can successfully invade hepatocytes. Abs can block extravasation and infection of hepatocytes. (Ci) Within each infected hepatocyte, the sporozoite transforms into a trophozoite stage, followed by cell growth and DNA replication during schizogony to ultimately generate tens of thousands of exoerythrocytic merozoites. Cytotoxic T cells and cytokine signaling can eliminate parasite-infected hepatocytes or eliminate the parasite within the hepatocyte, respectively. (ii) RAS and EARD GAPs arrest at the trophozoite/early schizont stage within hepatocytes. Because of this early developmental arrest, immunizations with RAS and EARD GAPs expose the immune system to a repertoire of Ags that is limited to sporozoites and early liver stages. (iii) LARC GAPs, in comparison, undergo nearly full schizogony and arrest late in development. LARC GAPs have large parasite biomass and express a broad repertoire of Ags encompassing sporozoite, early liver–stage, and late live–stage Ags, some of which are shared with BSs. During CPS vaccination (i), parasites undergo full schizogony within hepatocytes. Exoerythrocytic merozoites are released into circulation, and they invade RBCs, but chloroquine kills the parasite within RBCs. Immunizations with CPS primes the host with a broad repertoire of sporozoite, liver-stage, and BS Ags. (D) Immunizations with whole-sporozoite vaccines triggers potent cellular adaptive immunity to kill infected hepatocytes. Additionally, they drive a potent recruitment of myeloid cells and innate lymphoid cells into the liver. (E) Upon egress from the liver, merozoites initiate a 48-h cycle of infection, asexual replication, and destruction of RBCs. Some asexual parasites commit to gametocytogenesis and generate male and female gametocytes, which are then taken up by an Anopheles mosquito during a blood meal and the cycle continues. Immunization with RC whole-sporozoite vaccines, such as CPS or LARC GAP, induces both humoral and cellular immune responses against pre-erythrocytic– and erythrocytic-stage parasites.

Close modal

Repeated Plasmodium infections can result in partial, acquired immunity that first protects from severe malaria and subsequently reduces the incidence of clinical malaria by controlling BS parasite burden. Complete sterilizing, protective immunity to prevent reinfection has not been convincingly observed in a natural setting, implying that infected humans are either incapable of generating or of retaining a robust and durable immune response against the pre-erythrocytic stages of infection. Indeed, evidence from multiple laboratories indicates that BS-mediated inflammation negatively regulates humoral adaptive pre-erythrocytic immunity in rodent models of infection and in humans (7, 8).

P. falciparum infection is associated with the majority of malaria disease–associated mortality, and therefore it has been the primary focus of vaccine development efforts. Yet despite decades of research, why has it proven to be so difficult to develop an efficacious antimalarial vaccine? The answer lies in the extreme complexity and adaptive capabilities of the parasites. They have large genomes with over 5000 genes, with gene sequences showing significant divergence among strains of the same parasite species, particularly in P. falciparum. Gene expression occurs differentially as the parasite progresses through its life cycle, rendering Plasmodium a challenging target for traditional subunit vaccine approaches. Moreover, vaccination approaches that have targeted the symptomatic BS have met with limited success likely because of the complex Ag diversity and antigenic variation (9, 10). In contrast, the pre-erythrocytic stages of the parasite life cycle constitute an asymptomatic population bottleneck with a low parasite load, and targeting this stage can elicit both humoral immunity against sporozoites and cell-mediated immunity against infected hepatocytes that together can prevent onset of BS infection and disease (1116).

In this review, we discuss recent advances in the discovery, development, and immunology of pre-erythrocytic vaccines against malaria, with special focus on genetically attenuated, whole sporozoite–based vaccines.

Subunit malaria vaccines: Lessons from RTS,S.

The most clinically advanced vaccine against malaria is the RTS,S subunit vaccine (Mosquirix, Glaxo Smith Kline), which integrates a truncated form of the immunodominant P. falciparum sporozoite surface protein, termed circumsporozoite protein (CSP), into the licensed hepatitis B pediatric vaccine to generate anti-CSP humoral immunity. RTS,S showed promising short-term efficacy (4 wk) in malaria-naive individuals in early controlled human malaria infection (CHMI) challenge (17, 18) but showed only modest efficacy in malaria endemic regions. Short-term protection was mainly conferred by Abs against the central repeat region of CSP, but these Abs waned, and little durable protection was observed (17). The drop in efficacy was partially attributed to variability between CSP of the vaccine P. falciparum strain (3D7) and the P. falciparum CSP haplotypes prevalent in the endemic parasite strains (18). For a highly complex pathogen such as Plasmodium, subunit vaccine development must take advantage of advanced epitope identification to reveal more vulnerable target regions of parasite proteins, as highlighted by recent work on novel inhibitory mAbs against CSP (19, 20) alongside the incorporation of multiple Ags.

Replication-deficient, whole-sporozoite vaccines: Radiation-attenuated sporozoites and early liver stage–arresting, genetically attenuated parasites.

The moderate efficacy of RTS,S has rekindled interest in using whole sporozoites as immunogens for vaccination. Historic studies by R. Nussenzweig and colleagues (21) showed that immunization of mice with radiation-attenuated rodent malaria sporozoites could confer complete sterilizing protection against infectious sporozoite challenge, complementing earlier studies in birds (22). Radiation-attenuated sporozoites (RAS) infect hepatocytes, but the parasites then fail to undergo DNA replication and arrest in further development (Fig. 1Cii). In human trials, complete sterilizing protection was induced after immunization through the bites of more than 1000 mosquitoes that carried radiation-attenuated P. falciparum sporozoites (2325). Over the past decade, an injectable formulation of RAS, known as the vaccine PfSPZ (based on the P. falciparum NF54 strain), has been developed by Sanaria and has undergone significant clinical testing resulting in a shift from ineffectual s.c. and intradermal routes of immunization to efficacious direct venous administration (24, 26, 27). CHMI studies show that PfSPZ vaccination can generate durable (12–14 mo after the last immunization) protection (28, 29). Additionally, PfSPZ vaccination also engenders protection against CHMI with a different P. falciparum strain (29, 30) and protection, albeit less, against natural infection (31). These encouraging results have initiated further clinical trials with PfSPZ in Africa (32, 33). To date, nearly 2000 subjects have received multiple doses of the PfSPZ vaccine, indicating that whole-sporozoite immunization in humans is feasible and safe. Further improvement of efficacy might be achieved with optimization of immunization regimens as well as the inclusion of additional parasite strains into the PfSPZ formulation.

Intrinsic optimization of a whole-sporozoite vaccine is possible with the application of genetic engineering to whole-sporozoite immunogen design. Technical advances in genetic manipulation over the last decade have enhanced the efficiency and pace for generation of transgenic Plasmodium parasites, enabling targeted and reproducible parasite attenuation at a desired time point of liver stage development. Advantageously, genetic attenuation creates a homogenous, genetically identical population of sporozoites and eliminates the possibility of batch-to-batch variations associated with radiation/attenuation. Thus, once created, genetically attenuated parasites (GAPs) do not require any further downstream attenuation procedures.

The first-generation GAP were early liver stage–arresting, replication-deficient (EARD) GAP. These GAPs successfully invade hepatocytes and transform into liver-stage trophozoites but do not, or only partially, initiate schizogony and DNA replication (Fig. 1Cii). The gene candidates for EARD GAP generation were identified by comparing transcriptomes of sporozoites derived from mosquito salivary glands versus those derived from midgut oocysts (34, 35) to identify genes that were upregulated in infective sporozoites, and as such, might play important roles in sporozoite infection of the mammalian host. Initial studies that explored single or double gene deletions of upregulated in infective sporozoite genes provided proof of concept that they were promising targets for attenuation (3640). For example, deletion of three critical genes, the two infection-related 6-cys family proteins, P36 and P52, and the RNA stabilizing protein SAP1/SLARP in the rodent malaria model P. yoelii generated an EARD GAP triple knockout (GAP3KO) parasite (36, 4144). P. yoelii GAP3KO showed complete attenuation in mice, and immunization with GAP3KO conferred complete protection against an infectious P. yoelii challenge for up to 6 mo (45). These promising results led to the generation of a homologous P. falciparum EARD GAP3KO that did not show any breakthroughs in human liver–chimeric mice (46) or in humans (45), indicating that GAP3KO is safe for use in further clinical studies. A follow-up clinical study is currently ongoing (https://clinicaltrials.gov/ct2/show/NCT03168854?term=GAP&cond=Malaria%2CFalciparum&rank=2) to test the efficacy of the EARD P. falciparum GAP3KO in CHMI studies. In addition to P. falciparum GAP3KO, a further promising EARD vaccine candidate is a P. falciparum GAP carrying the double gene deletion of another 6-cys family, B9, and SAP1 (47), which is currently being studied in clinical trials. Thus, RAS and EARDs have reignited interest in live, attenuated, whole-sporozoite vaccines and have provided impetus to design a next-generation, whole-sporozoite vaccine.

Replication-competent, whole-sporozoite vaccines: Chloroquine prophylaxis and sporozoites and late liver–stage-arresting GAPs.

A replication-competent (RC) parasite vaccine is administered as sporozoite forms, but, following hepatocyte infection, replicates its genetic material and undergoes liver stage growth, thereby significantly expanding parasite biomass and Ag repertoire. Such a whole-parasite vaccine has the potential to elicit robust Ab responses against sporozoite Ags and robust T cell responses against liver stage Ags. The proof of concept for high efficacy potential of RC sporozoites comes from vaccination studies in humans using chloroquine prophylaxis and sporozoites (CPS), also called, more broadly, infection-treatment immunization. CPS yields the normal invasion of hepatocytes by sporozoites, complete liver–stage development and the release of infectious merozoites into the blood stream that go on to infect RBCs. However, unlike natural infection, in CPS immunizations, the intraerythrocytic parasites are eliminated in this first replication cycle because of the presence of the drug chloroquine (Fig. 1Ci). CPS immunizations were initially carried out in human subjects by infectious mosquito bite (48) and more recently with cryopreserved sporozoites (4951). Strikingly, the immunizing dose required to engender complete sterile protection against homologous CHMI was approximately one 10th to one 20th of that required with RAS and protection lasted for up to 2.5 y (51). However, it is important to note that immunizations with CPS so far protect poorly against heterologous strain CHMI (52, 53). This could be due to the low-sporozoite doses given in CPS immunization, which, although sufficient to protect against homologous challenge, might be insufficient to protect against heterologous challenge (49). As such, CPS provides the strongest evidence in humans that RC parasites that complete liver–stage development engender sterile protection against malaria at lower immunization doses when compared with replication-deficient (RD) parasites such as RAS. Thus, a late liver stage–arresting, RC (LARC) GAP that undergoes near complete intrahepatocytic development to increase parasite biomass and diversity before ceasing development might be a superior vaccine than EARD GAP (Fig. 1Ciii).

LARCs have already been created in rodent malaria parasites by deleting genes encoding enzymes catalyzing fatty acid biosynthesis, including FabB/F. Fabb/f LARC parasites invade hepatocytes, replicate their genomes, and undergo near-complete schizogony but then arrest in maturation and do not form infectious exoerythrocytic merozoites (54). Complete durable and sterile protection was observed in fabb/f LARC-immunized mice, correlating with more breadth of the adaptive immune response to pre-erythrocytic stages, and a significantly lower dose of sporozoites was required when compared with EARD GAP. However, the fabb/f LARC could not be pursued in P. falciparum because fatty acid biosynthesis in this parasite is essential for sporozoite development (55). Based on late liver–stage transcriptome data, additional genes have been explored to generate new LARC GAPs (5659). We have recently generated a novel LARC GAP, the P. yoelii lisp2/mei2 LARC that completely arrests late in liver development and elicits durable sterile protection in immunized mice (60). However, to date, no LARC has been generated in P. falciparum. Yet, given the advances made in the generation of LARCs in rodent malaria models and the parallel advances seen in the genetic manipulation in P. falciparum, it is likely that progress will be made on generating this type of attenuated parasite for human vaccination.

RC versus RD whole-sporozoite vaccines.

RC whole-parasite vaccines constitute a promising next generation of attenuated parasite vaccines because of the following: 1) they express a larger repertoire of parasite Ags than RD parasites and therefore elicit broader cell-mediated and humoral immune responses; 2) they persist longer in the liver (days) compared with RD parasites and thereby increase the probability of Ag presentation and T cell priming; 3) they express a subset of BS Ags that can generate Abs against infected RBCs and thereby elicit stage-transcending protection against liver stages and BSs; and 4) they confer sterile protection at a dose of immunogen that is approximately 10- to 20-fold lower than RD parasite immunization and thus should require fewer sporozoites immunizations and fewer boosts to achieve sterile protection (Fig. 2). A persistent challenge in pursuing RC parasite vaccines will be the issue of safety, as the late liver–stage arrest-dependent attenuation might be difficult to achieve at a level that completely prevents transition of some parasites to BS infection.

FIGURE 2.

Distinct Ag repertoires of RAS, EARD GAP, LARC GAP, and CPS. (A) A schematic representation of how Plasmodium parasites undergo stage-specific gene expression as development proceeds from the sporozoite stage (red) to the liver stages (green) to the BS (blue). The yellow area depicts Ags that overlap between sporozoites and early liver stages, and the area in orange represents Ags that overlap between late liver stages and BSs. (B) A comparison of the Ag repertoires of RAS/EARD GAP, LARC GAP, and CPS. RAS or EARD express sporozoite Ags, Ags that overlap between sporozoite and liver stage and early liver–stage Ags, whereas LARC GAPs express sporozoite Ags (Ags that overlap between sporozoite and liver stage) and early liver–stage Ags, mid to late liver–stage Ags, and some Ags are shared with BS. CPS, in addition to the Ags expressed by LARC GAPs, likely also express some additional BS Ags as merozoites form, egress from the liver, infect RBCs, and initiate intraerythrocytic development. LARC GAPs and CPS, therefore, induce significantly more breadth in Ag repertoire as compared with RAS and EARD GAPs, thereby carrying the potential to elicit a broader diversity of Abs and Ag-specific CD8+ T cells.

FIGURE 2.

Distinct Ag repertoires of RAS, EARD GAP, LARC GAP, and CPS. (A) A schematic representation of how Plasmodium parasites undergo stage-specific gene expression as development proceeds from the sporozoite stage (red) to the liver stages (green) to the BS (blue). The yellow area depicts Ags that overlap between sporozoites and early liver stages, and the area in orange represents Ags that overlap between late liver stages and BSs. (B) A comparison of the Ag repertoires of RAS/EARD GAP, LARC GAP, and CPS. RAS or EARD express sporozoite Ags, Ags that overlap between sporozoite and liver stage and early liver–stage Ags, whereas LARC GAPs express sporozoite Ags (Ags that overlap between sporozoite and liver stage) and early liver–stage Ags, mid to late liver–stage Ags, and some Ags are shared with BS. CPS, in addition to the Ags expressed by LARC GAPs, likely also express some additional BS Ags as merozoites form, egress from the liver, infect RBCs, and initiate intraerythrocytic development. LARC GAPs and CPS, therefore, induce significantly more breadth in Ag repertoire as compared with RAS and EARD GAPs, thereby carrying the potential to elicit a broader diversity of Abs and Ag-specific CD8+ T cells.

Close modal

Whole-sporozoite immunization of mice, nonhuman primates, and humans engenders sterilizing pre-erythrocytic immunity (60). Moreover, RC whole-sporozoite vaccines engender superior immunity and engender greater protection at much lower doses than RD sporozoite vaccines (49, 61, 62). What drives the robust pre-erythrocytic immunity afforded by whole-sporozoite vaccines, and how can these immune responses be further enhanced to improve vaccine efficacy? GAPs offer a unique opportunity to rationally manipulate vaccine candidates to enhance their immunogenicity and efficacy while maintaining safety. Thus, identification of the protective innate and adaptive immune mechanisms after whole-sporozoite vaccination is critical for further refinement and improvement of both EARD and LARC GAPs.

Innate immunity to pre-erythrocytic infection.

Innate immunity serves as the first line of immune defense and is critical to shaping the adaptive immune response to infection. Historically, adjuvants targeting innate immune pathways and cells have been used to enhance vaccine efficacy. Yet, the effects of the innate immune responses on pre-erythrocytic infection and their impact on adaptive immunity remain woefully understudied. In particular, how does the contribution of innate immune signaling pathways alongside the following: 1) Ag presentation by hepatocytes, 2) Ag presentation by professional APCs, and 3) immunoregulation by recruited leukocytes ultimately contribute to the generation of protective pre-erythrocytic immunity?

Intradermal administration of sporozoites or mosquito bite delivery promotes the recruitment of neutrophils, CD8+CD11c+ dendritic cells, and inflammatory monocytes at the site of injection in the skin and also within skin-draining lymph nodes (SDLN). Although recruited neutrophils are dispensable for the ensuing CD8 T cell response (63), CD8+CD11c+ dendritic cells are required to present sporozoites Ags to naive CD8+ T cells (64) in the SDLN. Similar to intradermal administration, multiple studies indicate that CD8+CD11c+ dendritic cells are necessary for the generation of an optimal memory CD8 T cell response after i.v. immunization of mice with RAS (65, 66). However, the full spectrum of recruited innate leukocytes after whole-sporozoite immunization has not been elucidated.

We and others have shown that both type I IFN (IFN-1) and type II IFN signaling pathways are induced during liver infection by wild-type parasites, LARCs, and, to a lesser extent, EARDs (6769). This IFN-1 response promotes the recruitment of myeloid cells as well as innate and adaptive lymphocytes into the liver (67, 69). Given that immunizations with LARCs engender superior protection when compared with EARD, it is tempting to speculate that this is due, in part, to the more pronounced liver-stage innate immune response observed after immunization with the former. However, the liver is tolerogenic, and multiple studies have also identified pathogenic roles for IFN-1 in the generation of immunity postinfection with hepatotropic pathogens. For example, in hepatitis B virus–infected livers, IFN-1 induction promotes the production of IDO1 to limit the activation of Ag-specific CD8 T cell responses (70). Future studies to examine the impact of the IFN-1 signaling cascade, and the contribution of leukocytes recruited to the liver, on the ensuing adaptive immune response are urgently needed.

Although wild-type Plasmodium–infected hepatocytes are, to a significant degree, protected from host cell death (71, 72), infection with RD sporozoites renders hepatocytes highly susceptible to apoptosis (71, 73). Outstanding issues to be addressed include the following: 1) the identification of the cell death signaling cascades induced after immunization with RD and RC sporozoites; 2) the innate immune cells that are likely recruited to phagocytose and cross-present released parasite Ags; and, last, 3) the impact of cell death on the education of adaptive immunity. Seminal work by Yatim and colleagues (74) indicates that the induction of inflammatory signaling in dying cells is critical for effective cross-priming of CD8 T cells. Immunization with LARC GAP clearly induces significant inflammatory signaling in the infected hepatocyte (67, 69, 75). However, it remains to be determined whether arrest of the developing parasite within the infected hepatocyte also promotes hepatocyte cell death and what the consequence of this death is for adaptive immunity against pre-erythrocytic infection.

Various studies have implicated γδ T cells as critical to sporozoite-engendered immunity. In RAS- and CPS-immunized humans, an increase in the frequency of γδ T cells in the blood was observed that correlated with protection (2729, 31, 49, 76). However, the manner in which γδ T cells influence pre-erythrocytic adaptive immunity is a matter of debate. Initial studies (77) observed that the adoptive transfer of a single γδ T cell clone from RAS-immunized mice into naive mice was sufficient to limit liver-stage development after sporozoite inoculation (77), implying an effector role. In contrast, recent work indicates that depletion of γδ T cells during immunization, but not at the time of challenge, abolishes RAS-engendered sterile protection in mice, implying a role in the priming of robust pre-erythrocytic adaptive immunity but no effector role (76). Of interest, during Plasmodium BS infection, γδ T cells are major producers of cytokines critical for myeloid cell recruitment, differentiation, and maturation (78). Thus, a role for a γδ T cell subset in the recruitment/maturation of APCs during pre-erythrocytic infection seems probable.

Cell-mediated adaptive immunity to pre-erythrocytic infection.

Robust pre-erythrocytic immunity is critically dependent on the generation of memory CD8 T cells because protection against a sporozoite challenge is severely compromised in whole-sporozoite–immunized mice depleted of CD8 T cells at the time of challenge (13, 79). RAS immunization drives the production of both central memory CD8 T cells and effector memory CD8 T cells (TEM). However, immunization with viral vectors bearing a Plasmodium protein generated large numbers of central memory T cells, that did not confer protection (80), in contrast to immunization strategies that induced high levels of TEM that correlated well with protection (61, 81). Finally, recent studies with RAS-immunized mice indicate that noncirculating, anti-Plasmodium liver resident memory CD8 T cells (TRM) are critical to the protection afforded by whole-sporozoite immunization (8284). Thus, two memory CD8 T cell populations, TEM and TRM, likely function in concert to mediate protective immunity of whole-sporozoite vaccines, with TRM playing the more dominant role in the elimination of infected hepatocytes. A thorough examination of the factors required to drive TRM generation, survival, and maintenance after whole-sporozoite immunization will be critical to improving whole-sporozoite vaccines.

Immunization via direct i.v. inoculation of whole-sporozoites affords greater protection from a sporozoite challenge than s.c. and intradermal inoculation (26, 61). Moreover, RC sporozoite immunization generates superior TEM than RD sporozoite immunization (61). Understanding how different anatomical sites contribute to the priming of an effective memory CD8 T cell response might explain these observations. Earlier studies indicate that priming in the SDLN and peripheral lymph nodes after RD RAS immunization is sufficient to engender a robust pre-erythrocytic immune response (85). However, given that RC sporozoite vaccines undergo further development in infected hepatocytes, it is especially important to determine whether LARC GAP immunization, in contrast to EARD GAP immunization, promotes larger populations of Plasmodium-specific TEM or TRM that not only recognize early liver–stage-infected hepatocytes, but also those infected with mid- to late-stage parasites. Specifically, does priming in the liver or the liver-draining lymph nodes contribute to a more protective TRM and/or TEM response after RC sporozoite immunization as compared with RD sporozoite immunization? To date, no studies have conclusively shown that memory CD8 T cells generated after immunization with RC sporozoite vaccines can eliminate mid liver–stage- to late liver–stage-infected hepatocytes, whereas memory CD8 T cells generated after RD sporozoite vaccines are limited to early liver–stage Plasmodium–infected hepatocytes.

The elimination of infected hepatocytes requires MHC class I expression, yet the contribution of different cytokines and cytolytic proteins to protection depends on the rodent Plasmodium strains and the strain of mouse used for experimentation (79, 86). These results likely indicate that memory CD8 T cell–mediated elimination of infected hepatocytes requires both cytokine production and direct killing by the release of cytolytic proteins such as perforin and granzyme B. Importantly, the contribution of CD8 T cells to protective immune responses in humans remains completely unknown. In nonhuman primates, protection correlates with the generation of memory CD8 T cells in the livers of immunized animals (26).

In contrast to the multitude of mouse studies detailing the roles of CD8 T cells in pre-erythrocytic immunity, the impact of CD4 T cells is less well documented. CD4 T cell depletion at the time of challenge has no impact on protection (37), but depletion of CD4 T cells during immunization does abrogate protection, indicating that CD4 T cells are critical to the education and the generation of the pre-erythrocytic memory CD8 T cell response (87). Moreover, the exact mechanisms by which CD4 T cells promote CD8 T cell memory after whole-sporozoite immunization have not been clearly elucidated. Studies in RAS-immunized mice indicate that CD4 T cell help is necessary for the expansion and survival of memory CD8 T cells (87), yet whether this is simply through the secretion of growth factors such as IL-2 or via licensing of APCs that cross-present Ag to naive CD8 T cells remains to be determined. Thus, considerably more research is required to fully elucidate the protective roles of CD4 cells following both EARD and LARC sporozoite vaccination.

Humoral adaptive immunity to pre-erythrocytic stages.

Successful vaccines against most infectious diseases rely on the generation of effective class-switched, affinity-matured Abs against the disease-causing pathogen (88). Early evidence for the role of Abs in pre-erythrocytic immunity comes from studies using RAS-immunized mice in which Abs against sporozoite surface proteins were protective against parasite challenge (89). CSP was believed to be the main target of these Abs. Whole-sporozoite vaccines have the potential to generate neutralizing Abs not only against CSP, but also against other sporozoite Ags. Interestingly, it has been shown that CPS immunization in humans elicited Abs not only to sporozoite Ags such as CSP and thrombospondin-related anonymous protein (TRAP), but also to early liver–stage Ags such as EXP1 and late liver–stage Ags such as LSA1, MSP1, and LISP2 (49).

Studies with rodent malaria models have shown that passive transfer of immune sera from LARC GAP-immunized mice to naive mice, followed by infected mosquito bite challenge, resulted in complete sterile protection, thereby giving direct evidence for the contribution of GAP-induced humoral immunity to protection (90). Interestingly, when protection in the same passive transfer model was compared between EARD and LARC-immunized mice, the sera from LARC GAP-immunized mice resulted in better protection (62), implying that LARC GAP-immunized mice generate a superior humoral response. Abs elicited after immunization with the LARC GAP were reactive to Ags on sporozoites, liver stages, and BSs (60, 62), showing that LARC elicit Abs to a more diverse repertoire of Ags.

The only preliminary evidence for the contribution of GAP-engendered humoral responses in humans has been generated from a phase I safety trial of the EARD GAP, P. falciparum GAP3KO. A single immunization with P. falciparum GAP3KO is sufficient to induce a potent humoral immune response against the immunodominant sporozoite surface protein CSP (45). Moreover, immune sera from these subjects blocked sporozoite invasion of hepatocytes in vitro and reduced liver-stage burden in vivo in humanized mice (45). Surprisingly, sera from subjects with the highest CSP titers exhibited the least protection, implying that Ab-mediated protection is unlikely to be due to CSP alone but also responses to non-CSP sporozoite and/or liver stage Ags. The ongoing efficacy trial with EARD P. falciparum GAP3KO using CHMI to determine protection will provide valuable insights into underlying questions such as how Ab profiles from vaccinated subjects correlate with protection.

The extensive rodent model and human studies with EARD sporozoites have demonstrated that whole-sporozoite immunization is feasible, safe, and efficacious. Immunization with RC sporozoite vaccines confers superior immunity in humans and mice and affords more durable protection. Studies to date suggest that RC sporozoite vaccines represent a promising path forward for the development of a highly efficacious malaria vaccine. However, the requirement for drug cover and compliance issues precludes CPS as a feasible immunization strategy for the billions of people living in malaria endemic regions. Thus, LARC GAPs represent the most likely whole-sporozoite vaccine moving forward. LARC GAPs elicit durable immunity, and because they are amenable to genetic manipulation, they can be iteratively improved to express diverse Ags including BS and gametocyte Ags. Excitingly, future iterations of LARC GAP could also be modified to include immunomodulatory elements, Ags from additional malaria parasite species, and other pathogens coendemic with malaria. Efforts to understand the enhanced immunogenicity of LARC GAP and delineate correlates of protection in rodent malaria models, but more importantly in humans, will be critical to the rationale creation of an efficacious P. falciparum LARC GAP. Extensive studies will be needed in rodent malaria models and with human whole parasite vaccine candidates in humanized mice to ensure their complete attenuation. Significant challenges remain for any type of whole-sporozoite vaccine, including large-scale manufacturing of sporozoites, effective cryopreservation of these sporozoites in liquid nitrogen, and vaccine storage and delivery. Yet these challenges ought to be addressed head on, as whole parasite vaccines constitute an extremely valuable path to developing new tools for the elimination of malaria.

We thank Dr. Ashley Vaughan for critical review of the manuscript.

This work was supported by the National Institutes of Health (Grants R01 AI114699 and R01 AI125706).

Abbreviations used in this article:

BS

blood stage

CHMI

controlled human malaria infection

CPS

chloroquine prophylaxis and sporozoite

CSP

circumsporozoite protein

EARD

early liver stage–arresting, replication-deficient

GAP

genetically attenuated parasite

GAP3KO

GAP triple knockout

IFN-1

type I IFN

LARC

late liver stage–arresting, RC

RAS

radiation-attenuated sporozoite

RC

replication-competent

RD

replication-deficient

SDLN

skin-draining lymph node

TEM

effector memory CD8 T cell

TRM

resident memory CD8 T cell

WHO

World Health Organization.

1
Vanderberg
,
J. P.
,
U.
Frevert
.
2004
.
Intravital microscopy demonstrating antibody-mediated immobilisation of Plasmodium berghei sporozoites injected into skin by mosquitoes.
Int. J. Parasitol.
34
:
991
996
.
2
Sinnis
,
P.
,
F.
Zavala
.
2012
.
The skin: where malaria infection and the host immune response begin.
Semin. Immunopathol.
34
:
787
792
.
3
Risco-Castillo
,
V.
,
S.
Topçu
,
C.
Marinach
,
G.
Manzoni
,
A. E.
Bigorgne
,
S.
Briquet
,
X.
Baudin
,
M.
Lebrun
,
J. F.
Dubremetz
,
O.
Silvie
.
2015
.
Malaria sporozoites traverse host cells within transient vacuoles.
Cell Host Microbe
18
:
593
603
.
4
Frevert
,
U.
,
S.
Engelmann
,
S.
Zougbédé
,
J.
Stange
,
B.
Ng
,
K.
Matuschewski
,
L.
Liebes
,
H.
Yee
.
2005
.
Intravital observation of Plasmodium berghei sporozoite infection of the liver.
PLoS Biol.
3
:
e192
.
5
Kaushansky
,
A.
,
S. H.
Kappe
.
2015
.
Selection and refinement: the malaria parasite’s infection and exploitation of host hepatocytes.
Curr. Opin. Microbiol.
26
:
71
78
.
6
Prudêncio
,
M.
,
M. M.
Mota
,
A. M.
Mendes
.
2011
.
A toolbox to study liver stage malaria.
Trends Parasitol.
27
:
565
574
.
7
Keitany
,
G. J.
,
K. S.
Kim
,
A. T.
Krishnamurty
,
B. D.
Hondowicz
,
W. O.
Hahn
,
N.
Dambrauskas
,
D. N.
Sather
,
A. M.
Vaughan
,
S. H. I.
Kappe
,
M.
Pepper
.
2016
.
Blood stage malaria disrupts humoral immunity to the pre-erythrocytic stage circumsporozoite protein.
Cell Rep.
17
:
3193
3205
.
8
Ryg-Cornejo
,
V.
,
L. J.
Ioannidis
,
A.
Ly
,
C. Y.
Chiu
,
J.
Tellier
,
D. L.
Hill
,
S. P.
Preston
,
M.
Pellegrini
,
D.
Yu
,
S. L.
Nutt
, et al
.
2016
.
Severe malaria infections impair germinal center responses by inhibiting T follicular helper cell differentiation.
Cell Rep.
14
:
68
81
.
9
Thera
,
M. A.
,
O. K.
Doumbo
,
D.
Coulibaly
,
M. B.
Laurens
,
A.
Ouattara
,
A. K.
Kone
,
A. B.
Guindo
,
K.
Traore
,
I.
Traore
,
B.
Kouriba
, et al
.
2011
.
A field trial to assess a blood-stage malaria vaccine.
N. Engl. J. Med.
365
:
1004
1013
.
10
Genton
,
B.
,
I.
Betuela
,
I.
Felger
,
F.
Al-Yaman
,
R. F.
Anders
,
A.
Saul
,
L.
Rare
,
M.
Baisor
,
K.
Lorry
,
G. V.
Brown
, et al
.
2002
.
A recombinant blood-stage malaria vaccine reduces Plasmodium falciparum density and exerts selective pressure on parasite populations in a phase 1-2b trial in Papua New Guinea.
J. Infect. Dis.
185
:
820
827
.
11
Vaughan
,
A. M.
,
S. H. I.
Kappe
.
2017
.
Malaria parasite liver infection and exoerythrocytic biology.
Cold Spring Harb. Perspect. Med.
7
:
a025486
.
12
Lindner
,
S. E.
,
J. L.
Miller
,
S. H.
Kappe
.
2012
.
Malaria parasite pre-erythrocytic infection: preparation meets opportunity.
Cell. Microbiol.
14
:
316
324
.
13
Schofield
,
L.
,
J.
Villaquiran
,
A.
Ferreira
,
H.
Schellekens
,
R.
Nussenzweig
,
V.
Nussenzweig
.
1987
.
Gamma interferon, CD8+ T cells and antibodies required for immunity to malaria sporozoites.
Nature
330
:
664
666
.
14
Fisher
,
C. R.
,
H. J.
Sutton
,
J. A.
Kaczmarski
,
H. A.
McNamara
,
B.
Clifton
,
J.
Mitchell
,
Y.
Cai
,
J. N.
Dups
,
N. J.
D’Arcy
,
M.
Singh
, et al
.
2017
.
T-dependent B cell responses to Plasmodium induce antibodies that form a high-avidity multivalent complex with the circumsporozoite protein.
PLoS Pathog.
13
:
e1006469
.
15
Van Braeckel-Budimir
,
N.
,
J. T.
Harty
.
2014
.
CD8 T-cell-mediated protection against liver-stage malaria: lessons from a mouse model.
Front. Microbiol.
5
:
272
.
16
Crompton
,
P. D.
,
J.
Moebius
,
S.
Portugal
,
M.
Waisberg
,
G.
Hart
,
L. S.
Garver
,
L. H.
Miller
,
C.
Barillas-Mury
,
S. K.
Pierce
.
2014
.
Malaria immunity in man and mosquito: insights into unsolved mysteries of a deadly infectious disease.
Annu. Rev. Immunol.
32
:
157
187
.
17
Olotu
,
A.
,
G.
Fegan
,
J.
Wambua
,
G.
Nyangweso
,
A.
Leach
,
M.
Lievens
,
D. C.
Kaslow
,
P.
Njuguna
,
K.
Marsh
,
P.
Bejon
.
2016
.
Seven-year efficacy of RTS,S/AS01 malaria vaccine among young African children.
N. Engl. J. Med.
374
:
2519
2529
.
18
Neafsey
,
D. E.
,
M.
Juraska
,
T.
Bedford
,
D.
Benkeser
,
C.
Valim
,
A.
Griggs
,
M.
Lievens
,
S.
Abdulla
,
S.
Adjei
,
T.
Agbenyega
, et al
.
2015
.
Genetic diversity and protective efficacy of the RTS,S/AS01 malaria vaccine.
N. Engl. J. Med.
373
:
2025
2037
.
19
Kisalu
,
N. K.
,
A. H.
Idris
,
C.
Weidle
,
Y.
Flores-Garcia
,
B. J.
Flynn
,
B. K.
Sack
,
S.
Murphy
,
A.
Schön
,
E.
Freire
,
J. R.
Francica
, et al
.
2018
.
A human monoclonal antibody prevents malaria infection by targeting a new site of vulnerability on the parasite.
Nat. Med.
24
:
408
416
.
20
Triller
,
G.
,
S. W.
Scally
,
G.
Costa
,
M.
Pissarev
,
C.
Kreschel
,
A.
Bosch
,
E.
Marois
,
B. K.
Sack
,
R.
Murugan
,
A. M.
Salman
, et al
.
2017
.
Natural parasite exposure induces protective human anti-malarial antibodies.
Immunity
47
:
1197
1209.e10
.
21
Nussenzweig
,
R. S.
,
J.
Vanderberg
,
H.
Most
,
C.
Orton
.
1967
.
Protective immunity produced by the injection of x-irradiated sporozoites of plasmodium berghei.
Nature
216
:
160
162
.
22
Richards
,
W. H.
1966
.
Active immunization of chicks against Plasmodium gallinaceum by inactivated homologous sporozoites and erythrocytic parasites.
Nature
212
:
1492
1494
.
23
Clyde
,
D. F.
,
H.
Most
,
V. C.
McCarthy
,
J. P.
Vanderberg
.
1973
.
Immunization of man against sporozite-induced falciparum malaria.
Am. J. Med. Sci.
266
:
169
177
.
24
Clyde
,
D. F.
1990
.
Immunity to falciparum and vivax malaria induced by irradiated sporozoites: a review of the University of Maryland studies, 1971-75.
Bull. World Health Organ.
68
(
Suppl.
):
9
12
.
25
Hoffman
,
S. L.
,
L. M.
Goh
,
T. C.
Luke
,
I.
Schneider
,
T. P.
Le
,
D. L.
Doolan
,
J.
Sacci
,
P.
de la Vega
,
M.
Dowler
,
C.
Paul
, et al
.
2002
.
Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites.
J. Infect. Dis.
185
:
1155
1164
.
26
Epstein
,
J. E.
,
K.
Tewari
,
K. E.
Lyke
,
B. K.
Sim
,
P. F.
Billingsley
,
M. B.
Laurens
,
A.
Gunasekera
,
S.
Chakravarty
,
E. R.
James
,
M.
Sedegah
, et al
.
2011
.
Live attenuated malaria vaccine designed to protect through hepatic CD8+ T cell immunity.
Science
334
:
475
480
.
27
Seder
,
R. A.
,
L. J.
Chang
,
M. E.
Enama
,
K. L.
Zephir
,
U. N.
Sarwar
,
I. J.
Gordon
,
L. A.
Holman
,
E. R.
James
,
P. F.
Billingsley
,
A.
Gunasekera
, et al
VRC 312 Study Team
.
2013
.
Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine.
Science
341
:
1359
1365
.
28
Ishizuka
,
A. S.
,
K. E.
Lyke
,
A.
DeZure
,
A. A.
Berry
,
T. L.
Richie
,
F. H.
Mendoza
,
M. E.
Enama
,
I. J.
Gordon
,
L. J.
Chang
,
U. N.
Sarwar
, et al
.
2016
.
Protection against malaria at 1 year and immune correlates following PfSPZ vaccination. [Published erratum appears in 2016 Nat. Med. 22: 692.]
Nat. Med.
22
:
614
623
.
29
Lyke
,
K. E.
,
A. S.
Ishizuka
,
A. A.
Berry
,
S.
Chakravarty
,
A.
DeZure
,
M. E.
Enama
,
E. R.
James
,
P. F.
Billingsley
,
A.
Gunasekera
,
A.
Manoj
, et al
.
2017
.
Attenuated PfSPZ vaccine induces strain-transcending T cells and durable protection against heterologous controlled human malaria infection.
Proc. Natl. Acad. Sci. USA
114
:
2711
2716
.
30
Epstein
,
J. E.
,
K. M.
Paolino
,
T. L.
Richie
,
M.
Sedegah
,
A.
Singer
,
A. J.
Ruben
,
S.
Chakravarty
,
A.
Stafford
,
R. C.
Ruck
,
A. G.
Eappen
, et al
.
2017
.
Protection against Plasmodium falciparum malaria by PfSPZ vaccine.
JCI Insight
2
:
e89154
.
31
Sissoko
,
M. S.
,
S. A.
Healy
,
A.
Katile
,
F.
Omaswa
,
I.
Zaidi
,
E. E.
Gabriel
,
B.
Kamate
,
Y.
Samake
,
M. A.
Guindo
,
A.
Dolo
, et al
.
2017
.
Safety and efficacy of PfSPZ Vaccine against Plasmodium falciparum via direct venous inoculation in healthy malaria-exposed adults in Mali: a randomised, double-blind phase 1 trial.
Lancet Infect. Dis.
17
:
498
509
.
32
Richie
,
T. L.
,
P. F.
Billingsley
,
B. K.
Sim
,
E. R.
James
,
S.
Chakravarty
,
J. E.
Epstein
,
K. E.
Lyke
,
B.
Mordmüller
,
P.
Alonso
,
P. E.
Duffy
, et al
.
2015
.
Progress with Plasmodium falciparum sporozoite (PfSPZ)-based malaria vaccines.
Vaccine
33
:
7452
7461
.
33
Olotu
,
A.
,
V.
Urbano
,
A.
Hamad
,
M.
Eka
,
M.
Chemba
,
E.
Nyakarungu
,
J.
Raso
,
E.
Eburi
,
D. O.
Mandumbi
,
D.
Hergott
, et al
.
2018
.
Advancing global health through development and clinical trials partnerships: a randomized, placebo-controlled, double-blind assessment of safety, tolerability, and immunogenicity of PfSPZ vaccine for malaria in healthy equatoguinean men.
Am. J. Trop. Med. Hyg.
98
:
308
318
.
34
Kappe
,
S. H.
,
M. J.
Gardner
,
S. M.
Brown
,
J.
Ross
,
K.
Matuschewski
,
J. M.
Ribeiro
,
J. H.
Adams
,
J.
Quackenbush
,
J.
Cho
,
D. J.
Carucci
, et al
.
2001
.
Exploring the transcriptome of the malaria sporozoite stage.
Proc. Natl. Acad. Sci. USA
98
:
9895
9900
.
35
Matuschewski
,
K.
,
J.
Ross
,
S. M.
Brown
,
K.
Kaiser
,
V.
Nussenzweig
,
S. H.
Kappe
.
2002
.
Infectivity-associated changes in the transcriptional repertoire of the malaria parasite sporozoite stage.
J. Biol. Chem.
277
:
41948
41953
.
36
Labaied
,
M.
,
N.
Camargo
,
S. H.
Kappe
.
2007
.
Depletion of the Plasmodium berghei thrombospondin-related sporozoite protein reveals a role in host cell entry by sporozoites.
Mol. Biochem. Parasitol.
153
:
158
166
.
37
Tarun
,
A. S.
,
R. F.
Dumpit
,
N.
Camargo
,
M.
Labaied
,
P.
Liu
,
A.
Takagi
,
R.
Wang
,
S. H.
Kappe
.
2007
.
Protracted sterile protection with Plasmodium yoelii pre-erythrocytic genetically attenuated parasite malaria vaccines is independent of significant liver-stage persistence and is mediated by CD8+ T cells.
J. Infect. Dis.
196
:
608
616
.
38
Jobe
,
O.
,
J.
Lumsden
,
A. K.
Mueller
,
J.
Williams
,
H.
Silva-Rivera
,
S. H.
Kappe
,
R. J.
Schwenk
,
K.
Matuschewski
,
U.
Krzych
.
2007
.
Genetically attenuated Plasmodium berghei liver stages induce sterile protracted protection that is mediated by major histocompatibility complex Class I-dependent interferon-gamma-producing CD8+ T cells.
J. Infect. Dis.
196
:
599
607
.
39
Mueller
,
A. K.
,
N.
Camargo
,
K.
Kaiser
,
C.
Andorfer
,
U.
Frevert
,
K.
Matuschewski
,
S. H.
Kappe
.
2005
.
Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface.
Proc. Natl. Acad. Sci. USA
102
:
3022
3027
.
40
Mueller
,
A. K.
,
M.
Labaied
,
S. H.
Kappe
,
K.
Matuschewski
.
2005
.
Genetically modified Plasmodium parasites as a protective experimental malaria vaccine. [Published erratum appears in 2007 Nature 446: 102.]
Nature
433
:
164
167
.
41
VanBuskirk
,
K. M.
,
M. T.
O’Neill
,
P.
De La Vega
,
A. G.
Maier
,
U.
Krzych
,
J.
Williams
,
M. G.
Dowler
,
J. B.
Sacci
Jr.
,
N.
Kangwanrangsan
,
T.
Tsuboi
, et al
.
2009
.
Preerythrocytic, live-attenuated Plasmodium falciparum vaccine candidates by design.
Proc. Natl. Acad. Sci. USA
106
:
13004
13009
.
42
van Schaijk
,
B. C.
,
C. J.
Janse
,
G. J.
van Gemert
,
M. R.
van Dijk
,
A.
Gego
,
J. F.
Franetich
,
M.
van de Vegte-Bolmer
,
S.
Yalaoui
,
O.
Silvie
,
S. L.
Hoffman
, et al
.
2008
.
Gene disruption of Plasmodium falciparum p52 results in attenuation of malaria liver stage development in cultured primary human hepatocytes.
PLoS One
3
:
e3549
.
43
Ishino
,
T.
,
Y.
Chinzei
,
M.
Yuda
.
2005
.
A Plasmodium sporozoite protein with a membrane attack complex domain is required for breaching the liver sinusoidal cell layer prior to hepatocyte infection.
Cell. Microbiol.
7
:
199
208
.
44
Douradinha
,
B.
,
M.
van Dijk
,
G. J.
van Gemert
,
S. M.
Khan
,
C. J.
Janse
,
A. P.
Waters
,
R. W.
Sauerwein
,
A. J.
Luty
,
B.
Silva-Santos
,
M. M.
Mota
,
S.
Epiphanio
.
2011
.
Immunization with genetically attenuated P52-deficient Plasmodium berghei sporozoites induces a long-lasting effector memory CD8+ T cell response in the liver.
J. Immune Based Ther. Vaccines
9
:
6
.
45
Kublin
,
J. G.
,
S. A.
Mikolajczak
,
B. K.
Sack
,
M. E.
Fishbaugher
,
A.
Seilie
,
L.
Shelton
,
T.
VonGoedert
,
M.
Firat
,
S.
Magee
,
E.
Fritzen
, et al
.
2017
.
Complete attenuation of genetically engineered Plasmodium falciparum sporozoites in human subjects.
Sci. Transl. Med.
9
:
eaad9099
.
46
Mikolajczak
,
S. A.
,
V.
Lakshmanan
,
M.
Fishbaugher
,
N.
Camargo
,
A.
Harupa
,
A.
Kaushansky
,
A. N.
Douglass
,
M.
Baldwin
,
J.
Healer
,
M.
O’Neill
, et al
.
2014
.
A next-generation genetically attenuated Plasmodium falciparum parasite created by triple gene deletion.
Mol. Ther.
22
:
1707
1715
.
47
van Schaijk
,
B. C.
,
I. H.
Ploemen
,
T.
Annoura
,
M. W.
Vos
,
L.
Foquet
,
G. J.
van Gemert
,
S.
Chevalley-Maurel
,
M.
van de Vegte-Bolmer
,
M.
Sajid
,
J. F.
Franetich
, et al
.
2014
.
A genetically attenuated malaria vaccine candidate based on P. falciparum b9/slarp gene-deficient sporozoites.
Elife
3
:
e03582
.
48
Roestenberg
,
M.
,
M.
McCall
,
J.
Hopman
,
J.
Wiersma
,
A. J.
Luty
,
G. J.
van Gemert
,
M.
van de Vegte-Bolmer
,
B.
van Schaijk
,
K.
Teelen
,
T.
Arens
, et al
.
2009
.
Protection against a malaria challenge by sporozoite inoculation.
N. Engl. J. Med.
361
:
468
477
.
49
Mordmüller
,
B.
,
G.
Surat
,
H.
Lagler
,
S.
Chakravarty
,
A. S.
Ishizuka
,
A.
Lalremruata
,
M.
Gmeiner
,
J. J.
Campo
,
M.
Esen
,
A. J.
Ruben
, et al
.
2017
.
Sterile protection against human malaria by chemoattenuated PfSPZ vaccine.
Nature
542
:
445
449
.
50
Roestenberg
,
M.
,
E. M.
Bijker
,
B. K.
Sim
,
P. F.
Billingsley
,
E. R.
James
,
G. J.
Bastiaens
,
A. C.
Teirlinck
,
A.
Scholzen
,
K.
Teelen
,
T.
Arens
, et al
.
2013
.
Controlled human malaria infections by intradermal injection of cryopreserved Plasmodium falciparum sporozoites.
Am. J. Trop. Med. Hyg.
88
:
5
13
.
51
Roestenberg
,
M.
,
A. C.
Teirlinck
,
M. B.
McCall
,
K.
Teelen
,
K. N.
Makamdop
,
J.
Wiersma
,
T.
Arens
,
P.
Beckers
,
G.
van Gemert
,
M.
van de Vegte-Bolmer
, et al
.
2011
.
Long-term protection against malaria after experimental sporozoite inoculation: an open-label follow-up study.
Lancet
377
:
1770
1776
.
52
Schats
,
R.
,
E. M.
Bijker
,
G. J.
van Gemert
,
W.
Graumans
,
M.
van de Vegte-Bolmer
,
L.
van Lieshout
,
M. C.
Haks
,
C. C.
Hermsen
,
A.
Scholzen
,
L. G.
Visser
,
R. W.
Sauerwein
.
2015
.
Heterologous protection against malaria after immunization with Plasmodium falciparum sporozoites.
PLoS One
10
:
e0124243
.
53
Walk
,
J.
,
I. J.
Reuling
,
M. C.
Behet
,
L.
Meerstein-Kessel
,
W.
Graumans
,
G. J.
van Gemert
,
R.
Siebelink-Stoter
,
M.
van de Vegte-Bolmer
,
T.
Janssen
,
K.
Teelen
, et al
.
2017
.
Modest heterologous protection after Plasmodium falciparum sporozoite immunization: a double-blind randomized controlled clinical trial.
BMC Med.
15
:
168
.
54
Vaughan
,
A. M.
,
M. T.
O’Neill
,
A. S.
Tarun
,
N.
Camargo
,
T. M.
Phuong
,
A. S.
Aly
,
A. F.
Cowman
,
S. H.
Kappe
.
2009
.
Type II fatty acid synthesis is essential only for malaria parasite late liver stage development.
Cell. Microbiol.
11
:
506
520
.
55
van Schaijk
,
B. C.
,
T. R.
Kumar
,
M. W.
Vos
,
A.
Richman
,
G. J.
van Gemert
,
T.
Li
,
A. G.
Eappen
,
K. C.
Williamson
,
B. J.
Morahan
,
M.
Fishbaugher
, et al
.
2014
.
Type II fatty acid biosynthesis is essential for Plasmodium falciparum sporozoite development in the midgut of Anopheles mosquitoes.
Eukaryot. Cell
13
:
550
559
.
56
Haussig
,
J. M.
,
K.
Matuschewski
,
T. W.
Kooij
.
2011
.
Inactivation of a Plasmodium apicoplast protein attenuates formation of liver merozoites.
Mol. Microbiol.
81
:
1511
1525
.
57
Dankwa
,
D. A.
,
M. J.
Davis
,
S. H. I.
Kappe
,
A. M.
Vaughan
.
2016
.
A Plasmodium yoelii Mei2-like RNA binding protein is essential for completion of liver stage schizogony.
Infect. Immun.
84
:
1336
1345
.
58
Orito
,
Y.
,
T.
Ishino
,
S.
Iwanaga
,
I.
Kaneko
,
T.
Kato
,
R.
Menard
,
Y.
Chinzei
,
M.
Yuda
.
2013
.
Liver-specific protein 2: a Plasmodium protein exported to the hepatocyte cytoplasm and required for merozoite formation.
Mol. Microbiol.
87
:
66
79
.
59
Annoura
,
T.
,
B. C.
van Schaijk
,
I. H.
Ploemen
,
M.
Sajid
,
J. W.
Lin
,
M. W.
Vos
,
A. G.
Dinmohamed
,
D. K.
Inaoka
,
S. R.
Rijpma
,
G. J.
van Gemert
, et al
.
2014
.
Two Plasmodium 6-Cys family-related proteins have distinct and critical roles in liver-stage development.
FASEB J.
28
:
2158
2170
.
60
Vaughan
,
A. M.
,
B. K.
Sack
,
D.
Dankwa
,
N.
Minkah
,
T.
Nguyen
,
H.
Cardamone
,
S. H. I.
Kappe
.
2018
.
A plasmodium parasite with complete late liver stage arrest protects against preerythrocytic and erythrocytic stage infection in mice.
Infect. Immun.
86
:
e00088-18
.
61
Butler
,
N. S.
,
N. W.
Schmidt
,
A. M.
Vaughan
,
A. S.
Aly
,
S. H.
Kappe
,
J. T.
Harty
.
2011
.
Superior antimalarial immunity after vaccination with late liver stage-arresting genetically attenuated parasites.
Cell Host Microbe
9
:
451
462
.
62
Sack
,
B. K.
,
G. J.
Keitany
,
A. M.
Vaughan
,
J. L.
Miller
,
R.
Wang
,
S. H.
Kappe
.
2015
.
Mechanisms of stage-transcending protection following immunization of mice with late liver stage-arresting genetically attenuated malaria parasites.
PLoS Pathog.
11
:
e1004855
.
63
Mac-Daniel
,
L.
,
M. R.
Buckwalter
,
M.
Berthet
,
Y.
Virk
,
K.
Yui
,
M. L.
Albert
,
P.
Gueirard
,
R.
Ménard
.
2014
.
Local immune response to injection of Plasmodium sporozoites into the skin.
J. Immunol.
193
:
1246
1257
.
64
Radtke
,
A. J.
,
W.
Kastenmüller
,
D. A.
Espinosa
,
M. Y.
Gerner
,
S. W.
Tse
,
P.
Sinnis
,
R. N.
Germain
,
F. P.
Zavala
,
I. A.
Cockburn
.
2015
.
Lymph-node resident CD8α+ dendritic cells capture antigens from migratory malaria sporozoites and induce CD8+ T cell responses.
PLoS Pathog.
11
:
e1004637
.
65
Lau
,
L. S.
,
D.
Fernandez-Ruiz
,
V.
Mollard
,
A.
Sturm
,
M. A.
Neller
,
A.
Cozijnsen
,
J. L.
Gregory
,
G. M.
Davey
,
C. M.
Jones
,
Y. H.
Lin
, et al
.
2014
.
CD8+ T cells from a novel T cell receptor transgenic mouse induce liver-stage immunity that can be boosted by blood-stage infection in rodent malaria.
PLoS Pathog.
10
:
e1004135
.
66
Montagna
,
G. N.
,
A.
Biswas
,
K.
Hildner
,
K.
Matuschewski
,
I. R.
Dunay
.
2015
.
Batf3 deficiency proves the pivotal role of CD8α+ dendritic cells in protection induced by vaccination with attenuated Plasmodium sporozoites.
Parasite Immunol.
37
:
533
543
.
67
Miller
,
J. L.
,
B. K.
Sack
,
M.
Baldwin
,
A. M.
Vaughan
,
S. H.
Kappe
.
2014
.
Interferon-mediated innate immune responses against malaria parasite liver stages.
Cell Rep.
7
:
436
447
.
68
Liehl
,
P.
,
P.
Meireles
,
I. S.
Albuquerque
,
M.
Pinkevych
,
F.
Baptista
,
M. M.
Mota
,
M. P.
Davenport
,
M.
Prudêncio
.
2015
.
Innate immunity induced by Plasmodium liver infection inhibits malaria reinfections.
Infect. Immun.
83
:
1172
1180
.
69
Liehl
,
P.
,
V.
Zuzarte-Luís
,
J.
Chan
,
T.
Zillinger
,
F.
Baptista
,
D.
Carapau
,
M.
Konert
,
K. K.
Hanson
,
C.
Carret
,
C.
Lassnig
, et al
.
2014
.
Host-cell sensors for Plasmodium activate innate immunity against liver-stage infection.
Nat. Med.
20
:
47
53
.
70
Ito
,
H.
,
T.
Ando
,
K.
Ando
,
T.
Ishikawa
,
K.
Saito
,
H.
Moriwaki
,
M.
Seishima
.
2014
.
Induction of hepatitis B virus surface antigen-specific cytotoxic T lymphocytes can be up-regulated by the inhibition of indoleamine 2, 3-dioxygenase activity.
Immunology
142
:
614
623
.
71
Kaushansky
,
A.
,
P. G.
Metzger
,
A. N.
Douglass
,
S. A.
Mikolajczak
,
V.
Lakshmanan
,
H. S.
Kain
,
S. H.
Kappe
.
2013
.
Malaria parasite liver stages render host hepatocytes susceptible to mitochondria-initiated apoptosis.
Cell Death Dis.
4
:
e762
.
72
van de Sand
,
C.
,
S.
Horstmann
,
A.
Schmidt
,
A.
Sturm
,
S.
Bolte
,
A.
Krueger
,
M.
Lütgehetmann
,
J. M.
Pollok
,
C.
Libert
,
V. T.
Heussler
.
2005
.
The liver stage of Plasmodium berghei inhibits host cell apoptosis.
Mol. Microbiol.
58
:
731
742
.
73
Leiriao
,
P.
,
M. M.
Mota
,
A.
Rodriguez
.
2005
.
Apoptotic Plasmodium-infected hepatocytes provide antigens to liver dendritic cells.
J. Infect. Dis.
191
:
1576
1581
.
74
Yatim
,
N.
,
H.
Jusforgues-Saklani
,
S.
Orozco
,
O.
Schulz
,
R.
Barreira da Silva
,
C.
Reis e Sousa
,
D. R.
Green
,
A.
Oberst
,
M. L.
Albert
.
2015
.
RIPK1 and NF-κB signaling in dying cells determines cross-priming of CD8+ T cells.
Science
350
:
328
334
.
75
Zheng
,
H.
,
Z.
Tan
,
T.
Zhou
,
F.
Zhu
,
Y.
Ding
,
T.
Liu
,
Y.
Wu
,
W.
Xu
.
2015
.
The TLR2 is activated by sporozoites and suppresses intrahepatic rodent malaria parasite development.
Sci. Rep.
5
:
18239
.
76
Zaidi
,
I.
,
H.
Diallo
,
S.
Conteh
,
Y.
Robbins
,
J.
Kolasny
,
S.
Orr-Gonzalez
,
D.
Carter
,
B.
Butler
,
L.
Lambert
,
E.
Brickley
, et al
.
2017
.
γδ T cells are required for the induction of sterile immunity during irradiated sporozoite vaccinations.
J. Immunol.
199
:
3781
3788
.
77
Tsuji
,
M.
,
P.
Mombaerts
,
L.
Lefrancois
,
R. S.
Nussenzweig
,
F.
Zavala
,
S.
Tonegawa
.
1994
.
Gamma delta T cells contribute to immunity against the liver stages of malaria in alpha beta T-cell-deficient mice.
Proc. Natl. Acad. Sci. USA
91
:
345
349
.
78
Mamedov
,
M. R.
,
A.
Scholzen
,
R. V.
Nair
,
K.
Cumnock
,
J. A.
Kenkel
,
J. H. M.
Oliveira
,
D. L.
Trujillo
,
N.
Saligrama
,
Y.
Zhang
,
F.
Rubelt
, et al
.
2018
.
A macrophage colony-stimulating-factor-producing gammadelta T cell subset prevents malarial parasitemic recurrence.
Immunity
48
:
350
363.e7
.
79
Schmidt
,
N. W.
,
N. S.
Butler
,
J. T.
Harty
.
2011
.
Plasmodium-host interactions directly influence the threshold of memory CD8 T cells required for protective immunity.
J. Immunol.
186
:
5873
5884
.
80
Reyes-Sandoval
,
A.
,
J. T.
Harty
,
S. M.
Todryk
.
2007
.
Viral vector vaccines make memory T cells against malaria.
Immunology
121
:
158
165
.
81
Schmidt
,
N. W.
,
N. S.
Butler
,
V. P.
Badovinac
,
J. T.
Harty
.
2010
.
Extreme CD8 T cell requirements for anti-malarial liver-stage immunity following immunization with radiation attenuated sporozoites.
PLoS Pathog.
6
:
e1000998
.
82
Tse
,
S. W.
,
I. A.
Cockburn
,
H.
Zhang
,
A. L.
Scott
,
F.
Zavala
.
2013
.
Unique transcriptional profile of liver-resident memory CD8+ T cells induced by immunization with malaria sporozoites.
Genes Immun.
14
:
302
309
.
83
Fernandez-Ruiz
,
D.
,
W. Y.
Ng
,
L. E.
Holz
,
J. Z.
Ma
,
A.
Zaid
,
Y. C.
Wong
,
L. S.
Lau
,
V.
Mollard
,
A.
Cozijnsen
,
N.
Collins
, et al
.
2016
.
Liver-resident memory CD8+ T cells form a front-line defense against malaria liver-stage infection.
Immunity
45
:
889
902
.
84
McNamara
,
H. A.
,
Y.
Cai
,
M. V.
Wagle
,
Y.
Sontani
,
C. M.
Roots
,
L. A.
Miosge
,
J. H.
O’Connor
,
H. J.
Sutton
,
V. V.
Ganusov
,
W. R.
Heath
, et al
.
2017
.
Up-regulation of LFA-1 allows liver-resident memory T cells to patrol and remain in the hepatic sinusoids.
Sci. Immunol.
2
:
eaaj1996
.
85
Obeid
,
M.
,
J. F.
Franetich
,
A.
Lorthiois
,
A.
Gego
,
A. C.
Grüner
,
M.
Tefit
,
C.
Boucheix
,
G.
Snounou
,
D.
Mazier
.
2013
.
Skin-draining lymph node priming is sufficient to induce sterile immunity against pre-erythrocytic malaria.
EMBO Mol. Med.
5
:
250
263
.
86
Butler
,
N. S.
,
N. W.
Schmidt
,
J. T.
Harty
.
2010
.
Differential effector pathways regulate memory CD8 T cell immunity against Plasmodium berghei versus P. yoelii sporozoites.
J. Immunol.
184
:
2528
2538
.
87
Overstreet
,
M. G.
,
Y. C.
Chen
,
I. A.
Cockburn
,
S. W.
Tse
,
F.
Zavala
.
2011
.
CD4+ T cells modulate expansion and survival but not functional properties of effector and memory CD8+ T cells induced by malaria sporozoites.
PLoS One
6
:
e15948
.
88
Sarkander
,
J.
,
S.
Hojyo
,
K.
Tokoyoda
.
2016
.
Vaccination to gain humoral immune memory.
Clin. Transl. Immunology
5
:
e120
.
89
Nussenzweig
,
R. S.
,
J. P.
Vanderberg
,
Y.
Sanabria
,
H.
Most
.
1972
.
Plasmodium berghei: accelerated clearance of sporozoites from blood as part of immune-mechanism in mice.
Exp. Parasitol.
31
:
88
97
.
90
Keitany
,
G. J.
,
B.
Sack
,
H.
Smithers
,
L.
Chen
,
I. K.
Jang
,
L.
Sebastian
,
M.
Gupta
,
D. N.
Sather
,
M.
Vignali
,
A. M.
Vaughan
, et al
.
2014
.
Immunization of mice with live-attenuated late liver stage-arresting Plasmodium yoelii parasites generates protective antibody responses to preerythrocytic stages of malaria.
Infect. Immun.
82
:
5143
5153
.

The authors have no financial conflicts of interest.