TLR1 and TLR6 are currently regarded as auxiliary receptors, which both form heterodimers with TLR2 in the plasma membrane (Kawai and Akira, 2010; Oliveira-Nascimento et al., 2012). These heterodimers of both TLR2/1 and TLR2/6 recognize a broad spectrum of different PAMPs, in particular multiple diacyl lipopeptides of various infectious agents such as viruses, bacteria, fungi, and parasites (Oliveira-Nascimento et al., 2012; De Almeda et al., 2013; Misch et al., 2013; Zhang et al., 2013). PAMPs recognition then activates a cascade of downstream reactions eventually leading to innate and adaptive immune responses directed against pathogens and, also, to protection from those adverse processes induced by host responses to infections (Kawai and Akira, 2010; Oliveira-Nascimento et al., 2012). In human malaria with *P. falciparum*, the TLR2/1 and TLR2/6 complexes recognize specific components of the glycosylphosphatidylinositols (GPIs), that are regarded as major factors contributing to malaria pathogenesis (Krishnegowda et al., 2005). Thus, TLR2/1 recognizes intact 3 acyl-GPIs and TLR2/6 the sn-2-lyso-GPI components (Krishnegowda et al., 2005). Remarkably, expression of *Tlr6* and *Tlr1*, but not that of *Tlr2*, *Tlr7*, and *Tlr8*, are under epigenetic control, since blood-stage infections of *P. chabaudi* induce alterations of the DNA methylation status of the promoters of *Tlr1* and *Tlr6* genes (Al-Quraishy et al., 2013). This malaria-induced epigenetic finetuning of *Tlr6* and *Tlr1* in the liver may be an important initial step of the host response against *Plasmodium* blood stages. Since DNA methylation is stable (Bird, 2002), it may be further suspected that epigenetic fine tuning of *Tlr6* and *Tlr1* may contribute to memory against homolog re-infections.

In contrast to *Tlr1* and *Tlr6*, the DNA methylation status of the *Tlr8* gene promoter is not affected by *P. chabaudi* infections at all. The function of TLR8, intracellularly localized in endosomal membranes, are not yet well understood (Cervantes et al., 2012; Kesar and Odin, 2014). TLR8 has been shown to sense singlestranded RNA of viral origin and bacterial RNA as well as to induce NFκB-dependent cytokines and type I IFNs. However, increasing information suggests that TLR8, possibly in cross-talk with TLR7 (Wang et al., 2006), is involved in the generation of autoimmunity (Krieg and Vollmer, 2007; DeMaria et al., 2010). Remarkably, dysregulations of *Tlr8* and *Tlr6* occur, when *P. chabaudi* infections are induced by testosterone to take a lethal outcome in female mice (Al-Quraishy et al., 2014).

#### **ACUTE PHASE RESPONSE**

An APR is experimentally inducible by blood-stage malaria, without any preceding pre-erythrocytic development of parasites in the liver, in humans and model systems. When in human volunteers an APR is induced by injecting *P. falciparum*-infected RBCs, serum levels of α1-acid glycoprotein (AGP) peak within 1 week (Klainer et al., 1968). AGP inhibits *in vitro* growth of *P. falciparum* by more than 80% at a concentration of 2.5– 3.0 mg/ml, which resembles those levels reached during a typical APR in humans (Friedman, 1983). Other APPs such as α1 antitrypsin, transferrin or α2-macroglobulin have been reported to be not as effective against *P. falciparum in vitro* (Friedman, 1983). Patients suffering from malaria reveal elevated serum CRP which binds to the patients' RBCs. These in turn loose their normal discoid-shape, increase membrane fluidity and hydrophobicity and decrease their effective complement-regulatory proteins (Ansar et al., 2009). In mice, blood-stage infections of both selfhealing *P. chabaudi* and *P. vinckei* as well as lethal *P. berghei* induce an APR characterized by increased serum levels of the major APP SAP (Balmer et al., 2000). In accordance, the mRNA levels of *Saa 1–3* in the liver increased by 65- to 80-fold at peak parasitemia of *P. chabaudi* infections (Krücken et al., 2005). By contrast, a reduced APR in association with a significant extension of primary parasitemia is induced in IL-6-deficient mice in response to blood-stage infections. This suggests that the APR induced by blood-stage infections contributes to an antiparasitic and/or immunomodulatory immune response (Balmer et al., 2000).

It is remarkable, however, that negative APPs, as, e.g., haptoglobin, PAI-1, and AP with potentially efficient anti-malaria activity, are decreased during the APR against blood-stage malaria. For instance, haptoglobin is malaria-protective, since it is toxic to *P. falciparum*-infected RBCs. Its main function is to bind hemoglobin released from erythrocytes and to support its elimination, thereby also preventing oxidative stress in the blood (Imrie et al., 2004). Hypohaptoglobinemia has been introduced as a biochemical and epidemiological marker of *P. falciparum* malaria (Mohapatra et al., 1999). In experimental *P. chabaudi* malaria, the *Pai-1* gene is downregulated in the liver. Since *Pai-1*-deficient mice cannot control parasitemia as good as wild type mice, PAI-1 has been suggested to be required for parasitemia control (Krücken et al., 2005). AP is also reduced at peak parasitemia during self-healing *P. chabaudi* infections (Wunderlich et al., 2005). AP exerts anti-inflammatory effects, as it is capable of dephosphorylating potentially deleterious molecules and of reducing inflammation and coagulopathy systemically (Pike et al., 2013). Obviously, the blood stages of the malaria parasites benefit from the down-regulation of negative APPs during an APR.

#### **INFLAMMATORY RESPONSE**

Course and outcome of both human and experimental bloodstage malaria critically depend on a finely tuned balance between both pro-inflammatory cytokines, as, e.g., IL-1β, TNFα, IFNγ, IL-6, and IL-12, and anti-inflammatory cytokines such as IL-4 and IL-10 (Bakir et al., 2011; Perkins et al., 2011).

In different experimental models, the liver has been shown to produce different cytokines in response to primary bloodstage malaria. However, there are differences between lethal and self-healing infections. In lethal *P. chabaudi* malaria, there occurs biphasic, two-wavy increase in serum IL-1β, TNFα, and IL-6 with the first wave peaking on day 1 *p.i.* and the second wave peaking at maximal parasitemia on day 8 *p.i.* (Wunderlich et al., 2012). In parallel, the liver biphasically produces mRNAs coding for IL-1β, TNFα, and IL-6 (Krücken et al., 2009). By contrast, the hepatic production of IFNγ mRNA reveals a lower and more uniform increase during precrisis. This pattern of mRNA production is totally reverted after protective vaccination of mice. Then, the liver produces a pronounced biphasic increase of IFNγ mRNA at higher levels, while the mRNA levels of IL-1β, TNFα, and IL-6 are lower and the biphasic production pattern has largely disappeared (Krücken et al., 2009). This indicates that protection against blood-stage malaria is associated with low production of IL-1β, TNFα, IL-6 and a high biphasic production IFNγ by the liver. Incidentally, a two-wavy production pattern, as well as its disappearance after protective vaccination, can be also found for the mRNA production of inducible nitric oxide synthase (iNOS), arginase, and different nuclear receptors including pregnane X receptor in the liver (Krücken et al., 2009).

In mice infected with a non-lethal strain of *P. yoelii*, the liver has been also shown to produce mRNAs of both proinflammatory cytokines, i.e., IFNγ, IL-12 p40, IL-6, and TNFα, and anti-inflammatory cytokines such as IL-4 and IL-10 (Bakir et al., 2011). Both pro- and anti-inflammatory cytokines are highly expressed during precrisis of blood-stage infections. It has been suggested that the pro-inflammatory cytokines induce the immune system to take action, while the anti-inflammatory cytokines counteract the high levels of inflammatory cytokines (Bakir et al., 2011).

The liver-produced cytokines are not only important for the local response in the liver, but also have presumably an impact for the systemic response to blood-stage malaria. For instance, a rather complex and dynamic interplay has been evidenced for IL-6. This pleiotropic cytokine is produced not only by HCs, but also by KCs and other liver cells. The severity of blood-stage malaria correlates with circulating IL-6 levels. Increased levels have been described in patients suffering from malaria caused by *P. falciparum* and *P. vivax* (Kern et al., 1989; Jason et al., 2001; Lyke et al., 2004; Robinson et al., 2009), often associated with polyclonal B cell activation (Donati et al., 2004). Conversely, decreasing IL-6 levels are reported to be associated with decreasing parasitemia (Sarthou et al., 1997) and hyperpyrexia (Seoh et al., 2003), as well as after anti-malarial treatment (Hugosson et al., 2006). In experimental murine malaria, it has been shown that IL-6 trans-signaling, rather than classic IL-6 signaling, contributes to malaria-induced lethality (Wunderlich et al., 2012). Indeed, approximately 50% of IL-6Rα-deficient mice survive an otherwise lethal *P. chabaudi* blood-stage malaria. However, the lethal outcome is restored, when IL-6 trans-signaling is induced by injecting mouse recombinant sIL-6Ra in the IL-6Rα-deficient mice. By contrast, inhibition of IL-6 trans-signaling through injecting recombinant sGP130Fc protein in wild type mice results in 40% survival (Wunderlich et al., 2012).

#### **PHAGOCYTIC ACTIVITY**

Intraerythrocytic stages of *Plasmodium* produce and store hemozoin in their phagosomes to detoxify the lytic ferriprotoporphyrin IX released during digestion of host hemoglobin by parasites (Olivier et al., 2014). Since ferriprotoporphyrin IX is also lytic for host cells including human RBCs (Orjih et al., 1988), the hemozoin is therefore cleared from the circulation as potentially dangerous waste material by phagocytosis of KCs to protect host cells.

On the other hand, KCs are also known to be endowed with the capacity to phagocytose and to remove senescent and aberrant erythrocytes including pRBCs from circulation (Otogawa et al., 2007; Lee et al., 2011) mediated by specific SRs (Terpstra and van Berkel, 2000). It has been therefore suspected that at least part of the intracellularly accumulated hemozoin in KCs is derived from internalized and decayed pRBCs. This view is supported by several findings. In rats with a high load of *P. berghei* bloodstage infections, KCs exhibit a markedly increased phagocytic activity, presumably due to an upregulation of scavenger activity for erythrophagocytosis (Nobes et al., 2002). Moreover, it has been shown in *P. berghei*-infected rats that the number of KCs is significantly increased and the phagocytic activity of KCs on a cell-by-cell basis is enhanced (Murthi et al., 2006). Furthermore, at peak parasitemia of *P. chabaudi*-infected mice, KCs are characterized by hyperplasia, and the massively enlarged KCs take on a "foamy" appearance detectable by light microscopy (Delic et al., 2010). These KCs contain, besides hemozoin, also regularly pRBCs inside the area of sectioned KCs thus supporting the view that KCs are indeed able to erythrophagocytose pRBCs and to destroy them in their phagosomes *in vivo*.

Several studies in non-KC macrophages have shown that ingestion of hemozoin by macrophages severely impairs macrophage activities upon diverse stimulations. For instance, hemozoin dramatically reduces the production of IL-6, but increases production of TNFα (Prada et al., 1995) and inhibits activities of NADPH-oxidase activity (Schwarzer and Arese, 1996) and protein kinase C (Schwarzer et al., 1993). Moreover, the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is inhibited (Schwarzer et al., 1993; Prada et al., 1996) and the expression of MHC II molecules, CD54 and CD11c is impaired (Schwarzer et al., 1998). However, hemozoin does not specifically affect expression of MHC I molecules, CD16 (low affinity Fc receptor for aggregated IgG), CD64 (high affinity receptor for IgG), CD11b (receptor for complement receptor iC3b), CD35 (receptor for complement components C3b and C4b), and CD36 (non-class-A SR; Schwarzer et al., 1998; Olivier et al., 2014). More recent data indicate that hemozoin can induce the production of IL-1β through the NOD-like containing pyrin 3 domain (NLRP3) inflammasome complex (Olivier et al., 2014). These data suggest that also *in vivo* activities of KCs may be negatively affected by ingested hemozoin. Indirect evidence for this view is provided by a magnetometric and electron microscopal study showing that ingestion and metabolism of *P. chabaudi*-infected RBCs by KCs *in vivo* decrease intracellular organelle motion in these KCs, which probably compromises host defense reactions (Bellows et al., 2011).

#### **"PROTECTIVE" AUTOIMMUNITY**

In humans, acquisition of natural immunity against bloodstage malaria is associated with the emergence of autoimmunity (Butcher, 2008). A number of studies with murine blood-stage malaria have substantiated the view that autoimmunity arises during blood-stage malaria and that the liver-inherent immune system is critically involved in the generation of this "antimalaria" autoimmunity. This is presumably not mediated by conventional T and B cells, but rather by special populations of hepatic T and B cells of innate immunity (Abo et al., 2012). For instance, *P. yoelii* infections have been reported to induce severe thymic atrophy in mice, which ultimately arrests production of conventional T cells (Abo et al., 2012). Concomitantly, however, extrathymic IL-2Rb+ CD3*int* T cells and its subset NK1.1− CD3*int* expand in the liver. Moreover, NK1.1− TCR*int* cells are present in the liver of athymic nude mice, which harbor neither conventional T cells nor NKT cells of thymic origin. When lymphocytes are isolated from the liver of athymic mice recovered from blood-stage malaria of non-lethal *P. yoelii*, these "immune" lymphocytes can confer protection from malaria to both irradiated euthymic and athymic mice (Mannoor et al., 2002). Furthermore, autoreactive extrathymic T cells have been described in the liver of malariainfected mice, which react with both HCs and RBCs of malariainfected mice (Abo et al., 2012).

In parallel with the expansion of extrathymic autoreactive T cells, the number of B220low B-1 cells is also increased in the liver and is highest around peak parasitemia in mice infected with nonlethal *P. yoelii* (Kanda et al., 2010; Abo et al., 2012). These B-1 cells produce autoantibodies against nucleus, ss- and dsDNA (Kanda et al., 2014). In accordance, serum levels of both IgM- and IgGtype autoantibodies against denatured DNA increased during non-lethal *P. yoelii* infections in mice, both peaking around peak parasitemia (Bakir et al., 2011). These immune responses resemble those found in autoimmune diseases (Bakir et al., 2011). *In vivo* depletion of autoreactive B-1 cells and subsequent infection with normally self-healing *P. yoelii* result in death of 56% of mice (Mannoor et al., 2013). By contrast, adoptive transfer of such auto-antibody secreting B cells prior to infecting with *P. yoelii* leads to later appearance and inhibition of parasitemia (Mannoor et al., 2013). Auto-antibodies secreting B220low B-1 cells have been suggested to target "abnormal self " cells, as, e.g., HCs and RBCs, in malaria-infected mice, and such autoantibody-labeled cells can be further processed and eliminated by macrophages and KCs (Kanda et al., 2014).

Previous authors have already suggested that autoimmunity plays a key regulatory role in protection from malaria (Jayawardena et al., 1979; Jarra, 1980). In accordance, it has been recently found that autoantibodies and sera from patients suffering from diverse autoimmune conditions, but not from malaria, inhibit the *in vitro* growth of *P. falciparum* in human RBCs (Bhatnagar et al., 2011; Brahimi et al., 2011). Especially that type of autoimmunity is apparently required for protection against bloodstage malaria (Daniel-Ribeiro, 2000), that is presumably directed against autoantigens and parasite-induced neo-autoantigens on the surface of *Plasmodium*-infected RBCs (Wunderlich et al., 1988b,c; Fontaine et al., 2012). It is therefore not surprising that surface membranes of *Plasmodium*-infected RBCs can be used for protective vaccination of malaria-susceptible mice, which raises their survival from 0 to more than 80% and decreases peak parasitemia by about 30% (Wunderlich et al., 1988a; Krücken et al., 2009).

Collectively, the currently available information supports the view that the generation of autoimmunity and/or the enhancement of already existing low level "natural" autoimmunity in the liver may be an integrative—if not the essential—part of protective immunity against blood-stage malaria (Abo et al., 2012). Malaria-activated TLR8, possibly in cross-talk with TLR7, has been speculated to be involved in the generation and/or surveillance of this "protective" autoimmunity (Al-Quraishy et al., 2014).

#### **LIVER INJURIES AND REPAIR**

Blood-stage malaria causes severe injuries of the liver. Humans infected with *P. falciparum* or *P. vivax* have been described to suffer from hepatic dysfunctions and malarial hepatitis with jaundice and raised liver enzyme levels in the blood (Ananad et al., 1992; Kochar et al., 2003; Nautiyal et al., 2005). Liver injuries have been also found *postmortem* in fatal human malaria, as, e.g., KC hyperplasia, hemozoin accumulation within KCs, livernecrosis, portal inflammation, steatosis, and cholestasis (Rupani and Amarapurkar, 2009). Similar histopathological injuries have been also observed in the liver of mice primarily infected with lethal *P. chabaudi* at peak parasitemia, besides increased serum levels of hepatic markers such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), bile acids, and bilirubin (Krücken et al., 2005; Delic et al., 2010). However, similar changes, though not as pronounced, also occur in the liver of mice when *P. chabaudi* infections take a self-healing course (Krücken et al., 2005; Delic et al., 2010).

Liver injuries are presumably not directly induced by the parasites, but largely indirectly as infection-induced overreactions of the liver-inherent immune system, as, e.g., increased oxidative stress induced by ROS/RNS produced during phagocytic activity of KCs, increased production of proinflammatory cytokines, increased release of perforin, granzymes, and lysosomal enzymes by LGL, autoreactive cytotoxic T cells, and "bystander killing" of liver cells due to cell-mediated cytotoxicity. For instance, hepatic CD1d-independent DX5+ T cells have been shown to cause "bystander killing" in the liver of *P. berghei*-infected mice (Adachi et al., 2004). Hepatotoxicity can be also induced by hepatic CD1drestricted NKT cells activated during blood-stage malaria through IL-12 secreted from KCs (Gonzalez-Aseguinolaza et al., 2000). Damages of liver cells have been suggested to be surveilled by DAMPs-activated TLRs (Adachi et al., 2001).

Even an apparent "non-immune" mechanism generating liver injury is very likely immune-mediated, as, for example, the almost doubling of the ammonia content in the blood plasma found at peak parasitemia during lethal *P. chabaudi* malaria (Delic et al., 2010). The increased systemic ammonia level signalizes severe local hepatic dysfunctions. Indeed, the liver metabolism is impaired, in particular the detoxifying capacity of the liver, evidenced as massive down-regulations of genes encoding enzymes involved in phase I–III metabolism (Krücken et al., 2005; Delic et al., 2010). However, similar changes in liver metabolism can be experimentally induced in mice only by injections of TNFα and IL-1β without any malaria infection (Kim et al., 2004, 2007). Increases in systemic ammonia levels presumably also cause dysfunctions of other organs. For instance, the brain is particularly sensitive to ammonia intoxication, which ultimately leads to hepatoencephalopathy (Butterworth, 2002; Häussinger and Schliess, 2008), that has been indeed reported to occur as a severe complication in *P. falciparum*-infected patients (Kochar et al., 2003; Whitten et al., 2011).

However, the liver also possess the capability for fast regeneration of destructed liver tissue. This capability also involves T cell-mediated repair mechanisms of the liver-inherent immune system. For instance, *P. chabaudi* blood-stage infections in mice have been described to induce an increase of liver-resident IL-17- and IL-22-producing CD8+ T cells (Mastelic et al., 2012). In contrast to IL-17 producing T cells, the IL-22 producing T cells very likely protect from the potentially lethal effects of liver damages during primary *P. chabaudi* infections. Further work is required to disentangle those host mechanisms in the liver, which mediate repair and healing of liver tissue injured during bloodstage malaria, and those, which contribute to liver dysfunctions and ultimately to multiple organ failure as the reason for malaria mortality (Delic et al., 2010).

#### **CONCLUDING REMARKS**

The importance of the liver-inherent immune system as part of the host defense against blood-stage malaria is increasingly recognized, though still too hesitatingly. The liver-inherent immune system is able to develop several different effector responses against malarial blood stages. Development and efficacy of these blood-stage effectors may be modulated by those immune responses which are previously elicited against the preerythrocytic liver stages of the malaria parasites (Klotz and Frevert, 2008) and, conversely, the latter may be even boosted by the blood-stage effectors (Ocana-Morgner et al., 2003; Lau et al., 2014).

Overreactions of the effector responses directed against bloodstage malaria presumably lead to severe inflammatory liver injuries having systemic impact. Such overreactions are puzzling since the liver-inherent immune system is also known for its capability to generate tolerance, a state of immune hyporesponsiveness. The "tolerogenic" milieu of the liver may be considered to be beneficial for blood-stage parasites of different *Plasmodium* species including *P. falciparum* and *P. chabaudi* to escape antimalaria effectors by intrahepatic sequestration (Medeiros et al., 2013; Brugat et al., 2014). Though intrahepatic sequestration mediated by CD36 has been reported to be fundamentally important for parasite growth *in vivo* (Fonager et al., 2012), parasite sequestration also occurs in other "non-tolerogenic" organs.

It is also puzzling how a tolerogenic hyporesponsive milieu as in the liver allows the generation of specific autoimmunity at all, that is presumably even an integrative—if not the essential part of protective immunity against blood-stage malaria. This Wunderlich et al. Liver in blood-stage malaria

autoimmunity is mediated by autoreactive extrathymic T cells and autoantibody-producing B-1 cells. One target of autoimmunity is the RBC, the host cell of the malaria blood stages. Remarkably, surface membranes of *Plasmodium*-infected RBCs, but not those of non-infected RBCs, can be used for protective vaccination of mice (Wunderlich et al., 1988a; Krücken et al., 2009). This raises survival of malaria-susceptible mice from 0 to more than 80%, decreases parasitemia and concomitantly attenuates overreactions of hepatic inflammation induced by blood-stage malaria of *P. chabaudi*. This vaccination model appears rather convenient to trace those immune and possible autoimmune mechanisms, which have to be activated and/or suppressed in the liver to generate efficient protection against blood-stage malaria. Such knowledge in turn will advance our understanding of how these mechanisms can be utilized for the design of an effective human anti-malaria vaccine.

#### **ACKNOWLEDGMENT**

To limit the number of citations, we have referred to reviews, in particular in the first part of this review. Supported by King saud University, National programme for Science and Technology (13- B101206-02).

#### **REFERENCES**

Duan, B., and Morel, L. (2006). Role of B-1a cells in autoimmunity. *Autoimmun. Rev.* 5, 403–408. doi: 10.1016/j.autrev.2005.10.007

B activation in mouse hepatocytes stimulated with lipopolysaccharide. *Infect. Immun.* 70, 3433–3442. doi: 10.1128/IAI.70.7.3433-3442.2002

immunity. *Am. J. Physiol. Regul. Integr. Comp. Physiol.* 3003, R1217–R1230. doi: 10.1152/ajpregu.00686.2011

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 26 August 2014; paper pending published: 30 September 2014; accepted: 06 October 2014; published online: 04 November 2014.*

*Citation: Wunderlich F, Al-Quraishy S and Dkhil MA (2014) Liver-inherent immune system: its role in blood-stage malaria. Front. Microbiol. 5:559. doi: 10.3389/fmicb. 2014.00559*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Microbiology.*

*Copyright © 2014 Wunderlich, Al-Quraishy and Dkhil. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Priming of CD8+ T cell responses to liver stage malaria parasite antigens

#### **Giampietro Corradin1\* and Jelena Levitskaya2**

1 Biochemistry Department, University of Lausanne, Epalinges, Switzerland

2 W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA

#### **Edited by:**

Ute Frevert, New York University School of Medicine, USA

#### **Reviewed by:**

John Harty, University of Iowa, USA Denise Doolan, Queensland Institute of Medical Research, Australia Dominique Mazier, University Pierre and Marie Curie, France

#### **\*Correspondence:**

Giampietro Corradin, Biochemistry Department, University of Lausanne, Chemin des Boveresses 157, 1066 Epalinges, Switzerland e-mail<EMAIL_ADDRESS>
While the role of malaria parasite-specific memory CD8+ T cells in the control of exoerythrocytic stages of malaria infection is well documented and generally accepted, a debate is still ongoing regarding both the identity of the anatomic site where the activation of naive pathogen-specific T cells is taking place and contribution of different antigenpresenting cells (APCs) into this process.Whereas some studies infer a role of professional APCs present in the lymph nodes draining the site of parasite injection by the mosquito, others argue in favor of the liver as a primary organ and hepatocytes as stimulators of naïve parasite-specific T cell responses. This review aims to critically analyze the current knowledge and outline new lines of research necessary to understand the induction of protective cellular immunity against the malaria parasite.

**Keywords: Plasmodium, malaria, liver stage antigens, antigen presentation toT cells, dendritic and liver cells as APC**

#### **INTRODUCTION**

It is currently accepted that priming of CD8+ T lymphocytes by antigen-presenting cells (APCs) takes place in the secondary lymphoid organs such as spleen and lymph nodes [reviewed in Ref. (1)]. Multi-photon-based intravital microscopy revealed that the first contact between naïve CD8+ T cells and APC takes place in the periphery of draining lymph nodes (DLN) shortly after infection and mainly occurs in the subcapsular sinus or the interfollicular regions enriched with pathogen-derived antigens (2, 3). Depending on the pathogen's nature, the rapid relocation of naïve T cells to the periphery of the draining lymph node can be either antigen-specific (2) or antigen-independent, associated with decreased local levels of chemokines and the drastic alteration of the lymph node architecture by the pathogen (3, 4). Data from mice infected with vesicular stomatitis virus demonstrated that, though CD169+ macrophages (5) residing in the subcapsular sinus were the major cell population bearing virus-derived antigens (2), dendritic cells (DCs) served as the primary APC triggering antigen-specific naïve CD8+ T cells. The ability of immature dendritic cells to acquire exogenous antigens followed by their proteolytic processing and presentation on the MHC class I molecules, commonly referred to as "crosspresentation," is believed to be the major requirement for the generation of primary antigen-specific CD8+ T cell responses against pathogens (6–9).

Upon the initial encounter of naïve T cells with APC, a heterogeneous progeny of antigen-specific CD8+ T cells including short-lived effector cells (SLEC) and memory precursor effector cells (MPEC) [reviewed in Ref. (10, 11)] is generated. It is still not clear whether the SLEC versus MPEC differentiation is enforced by the asymmetric segregation of transcription factors and protein degradation machinery already at the first cell division (12–14) or it reflects the differential exposure to inflammatory and co-stimulatory "help" signals received from APCs by antigenspecific CD8+ T cells during the expansion phase [reviewed in Ref. (10, 11)]. While generation of primary CD8+ T cell responses to non-inflammatory antigens requires CD4+ T cell help, induction of primary CD8+ T cells responses to *Listeria*, LCMV, and influenza virus is CD4+ T cell-independent and results from direct activation of APCs by the pathogen (15–17). Moreover, CD4+ T cell help can be replaced by the CD40 triggering on the DCs, which prime antigen-specific naïve CD8+ T cells (18, 19). Thus, the exact nature and requirements for "help" signals necessary for the initial triggering and subsequent expansion of primary antigen-specific CD8+ T cell responses vary among different pathogens and sites of primary infection. In this report, our objective is to present and discuss the published data regarding CD8+ T cell activation in *Plasmodium* infection, and suggest experiments to better understand the antigen presentation process.

Malaria infection is initiated through the bites by *Plasmodium*-carrying female Anopheles searching for blood to support egg development. As the mosquito probes the host environment under the skin for the presence of blood vessels, it injects salivary gland proteins both prior and during blood feeding to inhibit blood coagulation. Parasites deposited into the skin can also traverse surrounding cells and enter the circulation with subsequent infection of liver cells. Studies performed with parasites injected intradermally or intravenously show that the resulting liver parasite load is similar (20). In addition, transfer of parasites from the skin sites to DLN occurs (21).

Identification of the anatomical site and the type of APC, which orchestrate the induction of primary CD8+ T cell responses against a particular antigen, represents an essential step in rational design of CD8+ T cell-based vaccination strategies. Whereas the research on the effector phase of CD8+ T cell response against malaria has been quite extensive (22–25), a rather limited number

of studies attempted to dissect the issue of liver stage-specific CD8+ T cell priming in the infected host.

#### **ROLE OF DIFFERENT ORGANS IN ANTIGEN PRESENTATION**

In this respect, the study by Chakravarty et al. (21) appears to be one of the most comprehensive and systematic up to date. The authors concluded that extrahepatic lymphoid tissues, in particular the DLN and spleen are the most important sites contributing to the generation of the effector T-cell pool in the liver. In agreement with these data, Obeid and colleagues demonstrated that strictly subcutaneous immunization with irradiated sporozoites led to induction of sterile immunity against pre-erythrocytic malaria with T cell priming occurring in skin-draining lymph node (26). It was proposed that parasite-specific CD8+ T cell priming depends on cross-presentation of malaria antigens (21). This indicates that professional APC, rather than infected hepatocytes, trigger priming of naïve CD8+ T cells directed to liver stage antigens.

Several lines of experimental evidence were presented in support of these conclusions. Thus, IFNγ production by adoptively transferred circumsporozoite protein (CSP)-specific naïve transgenic T cells was first detected in the skin-DLN as early as on day 2 after mouse immunization by microinjection or mosquito bites, whereas no detectable T cell activation was detected in other organs including spleen. Hence, Chakravarty and co-authors suggested that these temporal differences in the onset of parasite-specific T cell activation could reflect the hierarchical order of T cell priming initiated in the DLN that could be followed by migration of primed CD8+ T cells to other organs, including the spleen and the liver. However, removal of lymph nodes draining the site of parasite injection prior to the adoptive transfer of parasite-specific CD8+ T cells, though resulted in a 60% reduction of activated CD8+ T cells in the liver, did not affect the frequencies of primed CD8+ T cells in the spleen where the first signs of T cells activation were documented only 24 h later than in DLN and at the same time point as in the liver. These data indicate that temporal differences in the onset of T cell activation used as a parameter for identification of the CD8+ T cell priming site should be carefully reconsidered in future studies and further strengthen the importance of the spleen as a site of induction of primary CD8+ T cell responses in animal models of the infection. The latter is in agreement with the data by Sano et al. (27) demonstrating that spleens of infected mice support priming of parasite-specific naïve CD8+ T cells following intravenous injection of sporozoites.

At the same time, several lines of evidence presented by Chakravarty and colleagues (21) do not firmly support the essential role of the spleen in the parasite-specific CD8+ T cell priming.

First, DCs isolated from the spleens 60 h after injection of sporozoites were unable to trigger proliferation of parasite-specific CD8+ T cells, whereas DCs isolated from the DLN efficiently induced T cell proliferation and, presumably, presented the antigen. Since no data with liver-resident DCs were generated, a direct role of intrahepatic professional APCs in priming of parasitespecific CD8+ T cells still needs to be addressed. In addition, as the first signs of activation of parasite-specific T cells in DLN were detected at day 2 post immunization, it is not completely clear whether DCs from DLN had a greater capacity to prime CD8+ T cells as compared to spleen and liver-resident DCs at time points earlier than 60 h.

Second, animals subjected to simultaneous lymphadenectomy and splenectomy prior to the adoptive transfer of CSP-specific CD8+ T cells followed by immunization with sporozoites and subsequent challenge with viable parasites 10 days later had similar load of parasites in the liver as non-immunized mice, indicating that either DLN or/and spleen are required for CSP-specific CD8+ T cell priming. At the same time, splenectomy alone did not affect inhibition of parasite development in the liver, prompting the authors to conclude, that DLNs are the priming site of protective CD8+ T cell responses.

Interestingly, as shown by Chakravarty and co-authors (21), removal of both DLNs and the spleen prior to immunization with sporozoites, though drastically reduced the activated T cell pool in the liver, failed to abrogate it completely, suggesting that at least a proportion of parasite-specific CD8+ T cells found in the liver had been primed outside the DLN and the spleen. These findings could reflect the process of parasite-specific CD8+ T cell triggering in the liver and define it as the organ essential for the parasite development. On the other hand, animals treated with FTY720, a drug, which inhibits lymphocyte egress from lymph nodes (28, 29), had substantially less IFN gamma producing parasite-specific CD8+ T cells at day 7 post injection with irradiated sporozoites. Based on this observation, the authors concluded that systemic distribution of CD8+ T cells, at least in part, contributes to the intrahepatic pool of parasite-specific CD8+ T cells (21). It still needs to be seen, if treatment with FTY720 (30) inhibits the development of "early-primed" parasite-specific CD8+ T cells in the liver and spleen, previously noted by the authors already 72 h after mosquito bite. In addition, effect of FTY720 on the protection of animals from subsequent challenge with live sporozoites has to be addressed in this model. Noteworthy, the time course of the parasite-specific clonal T cell activation in the lymph nodes, liver, and other organs is only slightly delayed (by 24 h) while it is known that activated T cells egress from the lymph nodes 4–5 days after antigen encounter (31, 32). The latter suggests that either activation of parasite-specific T cells may take place simultaneously in various organs, or unusually rapid egress from the lymph node after priming is an intrinsic feature of T cells in this specific experimental model.

#### **ROLE OF INFECTED HEPATOCYTES IN ANTIGEN PRESENTATION**

The role of infected hepatocytes in direct priming of naïve parasitespecific CD8+ T cells is still a subject of controversy. Early study by Renia et al. demonstrated that intrasplenic injection of infected hepatocytes induced protective T cell-mediated immunity against infection with *Plasmodium yoelii* and *P. berghei* sporozoites (33). Leiriao et al. demonstrated that apoptotic hepatocytes infected with irradiated sporozoites are phagocytosed by DCs and merely serve as a source of *Plasmodium* antigens for the initiation of the protective immune responses via cross-priming (34). In contrast, Renia and collaborators argued against apoptotic infected hepatocytes as a source of antigens and suggested that liver DCs could be activated upon uptake of parasite antigens directly from viable infected hepatocytes (35) as previously seen in other experimental models (36, 37). However, data from Chakravarty et al. implied that though cross-priming is required, it takes place in the DLNs and not in the liver (21). In agreement with these data, Jung et al. demonstrated that mice subjected to chemical depletion of CD11c+ DCs fail to induce CD8+ T cell responses to infection with *Plasmodium yoelii* (38). Neither of these studies considered hepatocytes as an APC subset capable of initiating the primary parasite-specific T cell responses.

A recent study by Balam et al. (39) focused on two questions: can infected hepatocytes directly prime naïve parasite-specific T cells and does stimulation of already primed CD8+ T cells protect mice against parasite challenge? Administration of CD8+ CSPspecific T cells but not an irrelevant T cell clone injected into TAP-deficient MHC class I mismatched recipient mice, simultaneously with infected hepatocytes bearing MHC haplotype relevant for parasite-specific T cells, resulted in 100% protection of mice from subsequent challenge with live sporozoites (39). As the observed protection was not due to a bystander effect or a continuous cytokine secretion by parasite-specific CD8+ T cells, these data demonstrate that infected hepatocytes are capable of presenting the antigen to CD8+ T cells, reactivating resting CSP-specific CD8+ T cells and inducing protection.

Importantly, more than 60% of naïve BALB/c mice injected with irradiated sporozoite-infected hepatocytes were also protected from subsequent live parasite challenge, suggesting that infected hepatocytes could contribute to the priming of endogenous naïve T cell. However, T cell depletion experiments are required to confirm that protection is T cell-mediated. Finally, to formally exclude contamination with other APC potentially present in the hepatocyte preparations and capable of presenting CSP and priming the naïve CD8+ T cells, isolation of pure hepatocyte population devoid of cells bearing markers of DCs, macrophages, and stellate cells should be done by flow cytometry using fluorescent transgenic parasites. On the other hand, arguing against the sole role of professional APC in priming of naïve immune responses to malaria parasites, mice depleted of DCs by treatment with cytochrome *c* were still protected from the challenge with live sporozoites in spite of significantly lower frequencies of endogenous parasite-specific T cells primed by the immunization with irradiated sporozoites (39). These data do not fully support the previously discussed role of dendritic cell function in induction of primary malaria liver stage-specific T cell responses (21, 38).

#### **OTHER CONSIDERATIONS**

The quality of hepatocytes as APCs capable of triggering T cells responses had been recently dissected by Ma et al. (40). It had been demonstrated that *P. berghei* and *P. falciparum* infected human hepatocytes retain largely unaltered expression of multiple molecules of the MHC class I pathway until very late stages of parasite development (40). Moreover, infected cells exhibited no obvious defects in the capacity to upregulate expression of different molecular components of the MHC class I machinery in response to pro-inflammatory lymphokines or trigger direct activation of allo-specific as well as peptide-specific human CD8+ T cells (40). At the same time, it is not known whether or not the characteristic features of professional APC believed to be important for efficient T-cell priming, i.e., co-stimulatory molecules B7.1 and B7.2 ("signal 2"), as well as production of cytokines essential for the survival and maintenance of primed T cells ("signal 3") are possessed by the primary human hepatocytes *in vivo* and/or induced upon infection.

Current literature dissecting the ability of primary hepatocytes to specifically prime naïve CD8+ T cells is scarce. Bertolino et al. demonstrated that purified primary murine hepatocytes were able to induce activation and proliferation of antigen-specific naive CD8+ T cells *in vitro*, even in the absence of exogenously added cytokines as well as CD80 and CD86 co-stimulatory molecules (41). Moreover, the magnitude of T cell proliferation induced by primary hepatocytes was comparable to that induced by DCs. Naïve T cell priming by hepatocytes did not require CD4+ T cell help and induced expression of early T cell activation markers and transient CD8+ T cell effector activity followed by rapid cell death of activated T cells. Thus, primary hepatocytes were able to prime naïve T cells but failed to sustain productive antigenspecific CD8+ T cell responses (41). In agreement with these data, *in vivo* experiments using endogenous expression of alloantigens under hepatocyte-specific promoters demonstrated that activation of primary T cells by hepatocytes as antigen-presenting cells leads to T cell apoptosis rather than formation of antigen-specific memory T cell pool (42–44). It was further demonstrated that T cells activated by hepatocytes died "by neglect" and lack of IL-2 and low expression of pro-survival genes due to insufficient costimulation during the priming phase (45). Hence, taking into account the inability of primary hepatocytes to provide appropriate co-stimulation during T cells priming along with the immunosuppressive microenvironment created by multiple subsets of the liver-resident APC [reviewed in Ref. (46, 47)], it may appear unlikely that hepatocytes infected with malaria parasites play a major role in the generation of effective parasite-specific CD8+ T cell memory responses. However, it does not preclude the possibility that CD8+ T cells specific to malaria antigens could be primed and activated, at least shortly, by hepatocytes supporting development of exo-erythrocytic forms. Indeed, given proper stimuli, such T cells can be rescued to full immunological competence and longer survival (48, 49). In the case of malaria, proper activation stimuli could be induced by *Plasmodium* infection leading to activation of numerous genes in hepatocytes (50, 51) including those involved in native immunity and antigen presentation. Since no transcriptional analysis has been performed in Kupffer cells traversed by sporozoites so far, it would be important to understand whether or not liver-resident macrophages change their immunomodulatory properties in the site of malaria infection.

At this point, a word of caution should be expressed to the fact that all animal studies discussed above were based on a single mouse strain, BALB/c, as well as a single CD8+ T cell epitope derived from the CSP. Future studies on the induction of primary T cell responses to exo-erythrocytic forms of malaria need to be extended to other protective CD8+ T cell epitopes including responses, which appear later in the liver stage by using either radiation attenuated (RAS) or genetically attenuated (GAS) sporozoites or sporozoites combined with chloroquine chemoprophylaxis (CPS) (52).

#### **FIELD STUDIES**

It is still unclear to what extend animal models dissecting induction of primary T cell responses to malaria as well as human studies involving vaccinated volunteers reflect the acquisition of natural cellular immune responses in malaria endemic areas. An acquisition of a sterile immune protection following immunization with RAS, GAS, or using CPS regime in animals and humans sharply contrasts with the situation in the field, where, in spite of frequent (up to 30 per month in certain areas) biting by infected mosquitoes (53, 54), no sterile protection is usually obtained in both adults and children in response to natural infection under drug treatment or intermittent preventive treatment (IPT). Several hypotheses could be proposed to explain this discrepancy: (1) sporozoite "charge" (and, as a result, supply of parasite antigens) is too small in the field as compared to that given under experimental conditions; (2) down-regulation of the parasite-specific CD8+ T cell responses by the content of mosquito salivary glands delivered together with sporozoites to the site of T cell priming; and (3) excessive and/or preceding induction of immune responses to salivary gland proteins. As for the latter, given the fact that only a fraction (0–25% depending on the seasons and location) of mosquitoes are infected (54–56), a memory T-cell pool specific for salivary gland antigens is most likely established prior to parasite infection. As a result, secondary T cell responses directed to mosquito antigens could be preferentially activated at the expense of parasite-specific T-cell activation via, for example, competition for IL-2, homeostatic niche, or by active secretion of inhibitory molecules (57–64). If this is the case, the efficacy of sporozoitebased pre-erythrocytic vaccines may turn out to be low in endemic areas due to even subtle contamination with the salivary gland proteins. Both the second and third hypotheses may explain the low frequency of parasite-specific helper and CD8+ T cell found in humans from malaria endemic regions, as well as the general failure [with the exception for a single donor so far (65)] to obtain stable human T-cell clones specific to malaria liver stage antigens.

#### **FINAL REMARKS**

In conclusion, existing experimental data obtained from animal models suggest that: (1) both DCs and hepatocytes can prime naïve malaria parasite-specific CD8+ T cells, at least those directed to epitopes derived from CSP and (2) either DCs or hepatocytes are sufficient to induce protective CSP-specific T cell responses if the parasite load is not excessive. Identification of the essential site for priming of malaria liver stage-directed CD8+ T cell responses of broader antigen specificity as well as mimicking the conditions of the natural exposure to the uninfected mosquito vector will pave the way for the optimal design of T cell-based vaccines. We hope that experimental approaches suggested above in the context of the reviewed original data (21, 27, 34, 39) will prompt further studies on the induction and maintenance of protective T cell responses against exo-erythrocytic stages of malaria infection.

#### **REFERENCES**

1. Zhang N, Bevan MJ. CD8(+) T cells: foot soldiers of the immune system. *Immunity* (2011) **35**:161–8. doi:10.1016/j.immuni.2011.07.010

64. Schneider BS, Soong L, Zeidner NS, Higgs S.*Aedes aegypti* salivary gland extracts modulate anti-viral and TH1/TH2 cytokine responses to sindbis virus infection. *Viral Immunol* (2004) **17**:565–73. doi:10.1089/vim.2004.17.565

65. Bonelo A, Valmori D, Triponez F, Tiercy JM, Mentha G, Oberholzer J, et al. Generation and characterization of malaria-specific human CD8(+) lymphocyte clones: effect of natural polymorphism on T cell recognition and endogenous cognate antigen presentation by liver cells. *Eur J Immunol* (2000) **30**:3079–88. doi:10.1002/1521-4141(200011)30:11<3079::AID-IMMU3079>3.0.CO;2-7

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 21 August 2014; paper pending published: 08 September 2014; accepted: 07 October 2014; published online: 05 November 2014.*

*Citation: Corradin G and Levitskaya J (2014) Priming of CD8*+ *T cell responses to liver stage malaria parasite antigens. Front. Immunol. 5:527. doi: 10.3389/fimmu.2014.00527*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Corradin and Levitskaya. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

**REVIEW ARTICLE** published: 17 November 2014 doi: 10.3389/fmicb.2014.00586

## Novel approaches to identify protective malaria vaccine candidates

#### *Wan Ni Chia1,2 , Yun Shan Goh1 and Laurent Rénia1,2 \**

1 Singapore Immunology Network, Agency for Science, Technology and Research, Singapore, Singapore

2 Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

#### *Edited by:*

Urszula Krzych, Walter Reed Army Institute of Research, USA

#### *Reviewed by:*

Julius Clemence Hafalla, London School of Hygiene and Tropical Medicine, UK Stasya Zarling, Walter Reed Army Institute of Research, USA

#### *\*Correspondence:*

Laurent Rénia, Singapore Immunology Network, Agency for Science, Technology and Research, 8a Biomedical Grove, Immunos, Biopolis, 138648 Singapore, Singapore e-mail: renia\_laurent@immunol. a-star.edu.sg

Efforts to develop vaccines against malaria have been the focus of substantial research activities for decades. Several categories of candidate vaccines are currently being developed for protection against malaria, based on antigens corresponding to the preerythrocytic, blood stage, or sexual stages of the parasite. Long lasting sterile protection from Plasmodium falciparum sporozoite challenge has been observed in human following vaccination with whole parasite formulations, clearly demonstrating that a protective immune response targeting predominantly the pre-erythrocytic stages can develop against malaria. However, most of vaccine candidates currently being investigated, which are mostly subunits vaccines, have not been able to induce substantial (>50%) protection thus far. This is due to the fact that the antigens responsible for protection against the different parasite stages are still yet to be known and relevant correlates of protection have remained elusive. For a vaccine to be developed in a timely manner, novel approaches are required. In this article, we review the novel approaches that have been developed to identify the antigens for the development of an effective malaria vaccine.

**Keywords: malaria, vaccine, library, antibodies, screen**

#### **INTRODUCTION**

Malaria is an infectious disease caused by the protozoan parasite *Plasmodium* and transmitted by the *Anopheles* mosquitoes. Malaria is a major public health problem, leading to high mortality and morbidity. Nearly half the world's population is at risk of contracting malaria (CDC, 2012). There are 207 million cases of clinical malaria and approximately 627,000 deaths in WHO (2012). There is currently no available vaccine. Age and host immune status are high risk factors for malaria, with young children under the age of five, pregnant women and travelers or migrants who lack immunity to the disease being most susceptible. Other risk factors include the infectivity and the transmission dynamics of the parasite strain (Doolan, 2011).

The *Plasmodium* parasite has a complex life cycle. Following an infected mosquito bite, sporozoites are inoculated into the dermis of the mammalian host (Vanderberg and Frevert, 2004; Amino et al., 2006). The sporozoites travel to the liver *via* the bloodstream and infect the hepatocytes (Amino et al., 2006). During this phase in the hepatocytes, sporozoites develop into schizonts over 2–14 days, depending on the species. Merosomes, merozoites containing vesicles, eventually bud out from infected hepatocytes to release merozoites, which then infect erythrocytes (Sturm et al., 2006; Baer et al., 2007).  Ookinetes, which results from gametocyte fusion, develop into oocysts in the midgut of the mosquito. Upon oocyst maturation, newly formed sporozoites migrate to the salivary gland of the mosquito, awaiting the next blood meal (Moorthy et al., 2004).

Symptoms of malaria include fever, headache, chills, sweating, and vomiting. Recurrent fever is one of the hallmarks of clinical malaria. This is a consequence of the release of malarial toxins into the bloodstream following repetitive rupture and re-invasion of erythrocytes. With disease progression, the red blood cell counts decreases and severe anemia might occur. Malarial infected red blood cells, such as those of *Plasmodium falciparum*, can also sequester in deep tissues, causing cerebral malaria, and organ failure. These severe pathologies can eventually lead to death.

#### **IMMUNE RESPONSES TO A MALARIA INFECTION**

Protective immunity against malaria requires a timely and coordinated interplay between the innate and adaptive immunity. This involves dendritic cells, NK cells, B cells, CD4+ and CD8+ T cells (Stevenson and Riley, 2004).

Sporozoite-specific antibodies can block sporozoites from migrating to the liver or from invading into hepatocytes, arresting disease progression (Rathore et al., 2005; Finney et al., 2014). Antibody-mediated immunity has been thought to be the central effectors of parasite clearance in the peripheral blood as MHC class I/II molecules are absent on the surface of infected red blood cells (Langhorne et al., 2008). The importance of antibodies was first demonstrated by Cohen et al. (1961), showing that passive transfer of immunoglobulins from immune adults into naïve, infected children resulted in rapid reductions of parasite density and resolution of clinical symptoms (Cohen et al., 1961). Merozoite-specific antibodies can prevent merozoites from invading erythrocytes (Michon et al., 2000; Dutta et al., 2005; Jiang et al., 2011) and mediate clearance of infected red blood cells by phagocytic cells *via* antibody-dependent cellular inhibition (Marsh and

Kinyanjui, 2006). Pathogen-specific antibodies secreted by B cells with CD4+ T helper cells enhancement are essential for clearance of parasitemia in the later stages of the infection (Langhorne et al., 2008).

In addition to the humoral arm of the adaptive immunity, cell-mediated immune responses are also crucial for protection against malaria.  Recent work has revealed an important role for IFNγ-secreting CD8+ T cells in preventing chronic *P. chabaudi* blood stage infection in mice (Horne-Debets et al., 2013). In human, sterile protection has been observed in experimental sporozoite challenge experiments following vaccination with whole sporozoites (Hoffman et al., 2002; Roestenberg et al., 2009; Seder et al., 2013). Both sporozoite-specific antibodies and T cells were induced.

#### **VACCINE DEVELOPMENT AGAINST MALARIA**

The rationale for vaccine development to protect against malaria stems from observations where naturally acquired immunity to malaria can protect individuals living in malaria-endemic regions against malaria in an age-dependent and exposuredependent manner (Gupta et al., 1999; Schofield and Mueller, 2006; Crompton et al., 2010). Although the protection is not sterilizing and is not always ensured for all chronically exposed individuals, passive transfer of sera from some chronically exposed individuals reduced strongly parasite levels in infected individuals (Cohen et al., 1961; Bouharoun-Tayoun et al., 1995). This demonstrated that antibodies can offer protection against the blood phase of the malarial infection.

More rationally, the typical Pasteur approach where attenuated parasites were used as vaccines has further demonstrated the feasibility of vaccination as protection against malaria. Immunization with irradiation-attenuated sporozoites has shown to confer sterile immunity against sporozoites challenge in animal models and in humans (Richards, 1966; Nussenzweig et al., 1967; Clyde et al., 1973; Rieckmann et al., 1974; Hoffman et al., 2002). The protective immune response involving antibodies and T cells was shown to target the pre-erythrocytic stages (Nussenzweig et al., 1967; Overstreet et al., 2008). In recent years, immunization with sporozoite or blood parasite under drug cover was also shown to confer strong protective immunity involving antibodies and T cells (Belnoue et al., 2002; Renia et al., 2006; Roestenberg et al., 2009; Friesen et al., 2010). Other approaches have used genetically attenuated parasites. Although these approaches have led to sterile immunity in mice (Mueller et al., 2005; Butler et al., 2012), they have yet to prove their efficacy in humans (Spring et al., 2013).

After decades of efforts spent on developing vaccines against malaria, no strong vaccine candidate has emerged as yet. Most vaccine development efforts are focused on only four antigens (WHO, 2012). The most clinically advanced candidate, RTS,S, conferred ∼50% protection from clinical *P. falciparum* malaria in children aged 5–17 months, and ∼30% protection in children aged 6–12 weeks (Agnandji et al., 2011, 2012). Vaccine efficacy

was undetectable 3 years after vaccination (Bejon et al., 2013). This level of protection is suboptimal and too low to achieve malaria eradication. No single molecular signature, key cellular determinant or immune mechanism of naturally acquired or vaccine-induced immunity has been unequivocally associated with protection (Offeddu et al., 2012). This impediment toward vaccine development for malaria is multi-factorial. The *Plasmodium* parasite has a diverse protein repertoire, and a complex life cycle involving both vertebrate and invertebrate hosts. There are antigenic polymorphisms and variations across parasite strains and species, and the parasite has developed sophisticated strategies to evade the host immunity.

With more reports of drug resistance and insecticide resistance in some endemic regions (Trape et al., 2011; Phyo et al., 2012; Ashley et al., 2014), management of the malarial disease has been increasingly difficult. Identification of new malarial antigens for vaccine development is critical. Here, we reviewed the different techniques that have been used to identify antigens recognized by antibodies induced during natural infections or after vaccinations with whole parasites.

#### **PRE-GENOMIC SCREENING METHODS FOR PROTECTIVE ANTIGENS**

The advent of new molecular biology techniques in the 1980s led to the discovery of many *Plasmodium* antigens. DNA libraries, consisting of cDNA or genomic DNA, were constructed in bacteriophage vectors for expression in *Escherichia coli* (Kemp et al., 1986). Antigens such as the circumsporozoite protein (CSP), the S-antigen, the ring erythrocyte surface antigen (RESA) were the first to be identified using mouse monoclonal antibodies (Ellis et al., 1983), sera from infected humans (Kemp et al., 1983; Stahl et al., 1984) or sera from infected mice or monkeys (Anders et al., 1984; Brown et al., 1984; Ardeshir et al., 1985). All the antigens discovered using these approaches were immunodominant or possessed immunodominant regions made of repeats (Kemp et al., 1987). Although immunodominant antigens induce very strong antibody responses, recent studies have shown that they did not offer adequate protection when tested as subunit vaccines in clinical trials (Spring et al., 2009; Doolan, 2011; Sheehy et al., 2012), suggesting that these antigens might be used by the *Plasmodium* parasite for immune evasion (Anders, 1986; Rodriguez et al., 2008). The CSP, an antigen of pre-erythrocytic stage malaria and the first malaria antigen to be cloned (Ellis et al., 1983), is one such example. Despite being immunodominant, the Phase III vaccine efficacy was suboptimal, with 55% reduction in the frequency of malaria episodes during the 12 months of follow-up in children 5–17 months of age and 35% in children 6–12 weeks old at first immunization (Agnandji et al., 2011). The protection waned to a mere 16.8% after 4 years of follow-up, indicating the lack of long lasting protection which declined with time and exposure to the parasite (Olotu et al., 2013). Similarly, while immunization with the apical membrane antigen-1 (AMA-1), an antigen expressed both at the pre-erythrocytic and erythrocytic stages and strongly immunodominant during the erythrocytic stage, elicited functional antibodies with *in vitro* inhibition against homologous blood parasites, volunteers developed parasitemia upon challenge with controlled human malaria infection (infection by mosquito

bites) without a significant reduction of peak parasitemia nor delay to patency (Spring et al., 2009).

The cDNA library approach is advantageous with a good representation of a stage-specific *Plasmodium* proteome, depending on the stage of the parasite at which the RNA was extracted from. However, the repertoire of the *Plasmodium* proteome the cDNA library represents is not entirely comprehensive and does not include antigens expressed by the parasite in the other stages of its life cycle. Nevertheless, it has successfully identified more than a hundred malaria antigens. Recently, Raj et al. (2014) created a *P. falciparum* blood stage cDNA library constructed in bacteriophage and expressed in *E. coli*. The antigen library was screened against sera from either infection-resistant or – susceptible chronically exposed individuals living in malaria-endemic regions. 2 out of 3 antigens identified were novel antigens. Antibody to one of the identified novel antigens, PfSEA-1, inhibited parasite growth and blocked schizont egress. Using the *P. berghei* ANKA strain ortholog of PfSEA-1, the authors demonstrated reduced parasitemia and longer survival in mice vaccinated with PbSEA-1. They further validated PfSEA-1 as a promising vaccine candidate through epidemiological studies, where anti-PfSEA-1 antibodies were strongly associated with lesser incidence of severe malaria in Tanzanian children and lower level of parasitemia in Kenyan adults.

#### **SCREENING METHODS TO IDENTIFY NEW ANTIGENS IN THE ERA FOLLOWING GENOME SEQUENCING**

At the turn of the 20th century, whole genomes of many *Plasmodium* strains were sequenced. These genome sequencing initiatives, by The Wellcome Trust Sanger Institute and The Institute for Genomic Research, have provided a wealth of data for the identification of protective antigens (Carlton et al., 2002; Gardner et al., 2002). In this review, we discuss the novel approaches (summarized in **Table 1**) to screen libraries of malarial antigen using sera from protected versus unprotected individuals to identify antigens associated with protection, in search for new protective antigens for vaccine development against malaria.

#### **NEW APPROACHES TO SEARCH FOR ANTIGENS INVOLVED IN PROTECTION**

The availability of whole genome sequences has enhanced the understanding of the parasite biology. Protein functions can be predicted and validated. Potential antigens could be screened for specific characteristics such as surface expression. Comparative genomics between different parasite strains allows identification of antigens that have limited variation, with the promise of greater vaccine coverage and hence efficacy in the field (Kooij et al., 2005).

Transcriptomics and proteomics has provided critical information on the expression profiles of malarial proteins during the parasite's life cycle (Florens et al., 2002; Le Roch et al., 2002; Bozdech et al., 2003; Hall et al., 2005) – the mosquito stage (Lindner et al., 2013), the liver stage (Tarun et al., 2008), and the sexual stage (Lasonder et al., 2002; Khan et al., 2005). *In silico* analysis of stage-specific transcription pattern identifies antigens that are differentially expressed for specific targeting to a specific stage of the parasite's life cycle and also antigens with conserved expression across the different stages for cross-stages targeting (Florens et al., 2002).

#### **SCREENING RECOMBINANT PROTEIN MICROARRAYS AGAINST IMMUNE SERA FROM VACCINATED HUMAN INDIVIDUALS**

Using the published *P. falciparum* and *P. vivax* genomes, a set of selected genes was targeted for protein expression using an *in vitro* transcription/translation system (Doolan et al., 2008; Tsuboi et al., 2008; Crompton et al., 2010; Barry et al., 2011; Trieu et al., 2011; Molina et al., 2012; Baum et al., 2013; Lu et al., 2014). These recombinant proteins were then printed onto microarray chips and probed with immune sera that were obtainedfrom human volunteers naturally exposed to malaria infections or immunized with radiation-attenuated sporozoites. Doolan et al. (2008) generated a protein microarray with 250 *P. falciparum* proteins and used it to profile antibody responses in sera from four groups of individuals: (1) protected, (2) non-protected individuals following vaccination with radiation-attenuated sporozoites, (3) partially protected individuals due to natural exposure, and (4) non-exposed individuals. The same group then expanded their library to include 1204 *P. falciparum* proteins (representing 23% of the *P. falciparum* genome; Crompton et al., 2010; Trieu et al., 2011; Baum et al., 2013) and also 91 *P. vivax* proteins (Molina et al., 2012). 22–29% of the screened proteins were found to be serodominant in immune sera, demonstrating a broad and varied response to many antigens. Antibody titres against current vaccine candidates such as CSP, atypical membrane antigen-1, liver stage antigen-3, merozoite surface protein-1 did not differ between immune and non-immune individuals (Crompton et al., 2010; Trieu et al., 2011).

Richards et al. (2013) expressed *P. falciparum* merozoite proteins, reported to have a role in erythrocyte invasion and/or localized on the merozoite surface or in the invasion organelles of the merozoites, in either bacterial or wheat germ cell-free expression systems. The recombinant proteins were tested for IgG immunoreactivity using sera from malaria-exposed Papua New Guinea children in ELISA. Forty six proteins were selected based on their ability to coat ELISA plates and their immunoreactivity. Sera from older children had higher immunoreactivity to most of the 46 proteins as compared to the younger children, indicating an acquisition of antibody responses with age, presumably due to prolonged exposure to the parasite. While merozoite surface proteins had higher seropositivity compared to the rhoptry and micronemal proteins, the antigens strongly correlated with protection were the rhoptry and micronemal proteins. This is consistent with other studies, suggesting that the key protective malarial antigens are the non-immunodominant antigens (Doolan et al., 2003, 2008; Crompton et al., 2010; Trieu et al., 2011; Baum et al., 2013). The authors proposed that a combinational vaccine consisting of non-immunodominant antigens, such as EBA, PfRh2 and PfRh4, is more likely to offer greater protection (Richards et al., 2013).

One of the main drawbacks with protein array is the extensive efforts needed to generate the soluble recombinant malarial antigens in the library (Doolan et al., 2008; Tsuboi et al., 2008). Cell-based expression systems have met with many difficulties. *P. falciparum*genes have a high A/T content and a substantial number

#### **Table 1 | Approaches taken to identify protective malarial antigens for vaccine development.**

of the genes encode stretches of repeat sequences (Gardner et al., 2002), hindering protein expression in cell-based expression system. Only 30% out of 1000 genes investigated by Mehlin et al. (2006) can be expressed in *E. coli* and a mere 6.3% of the proteins are soluble. The alternative is the wheat germ cell-free expression system. Tsuboi et al. (2008) were able to express 93 out of 124 *P. falciparum* genes as soluble proteins using the wheat germ cellfree system. Rui et al. (2011) reported greater immunogenicity of wheat germ proteins (as opposed to identical proteins produced

in *E. coli*) in mice. The wheat germ cell-free expression system could be more suitable for producing antigens for vaccine development.

#### **SCREENING CELL-ASSOCIATED ANTIGEN LIBRARIES AGAINST IMMUNE MOUSE SERA**

We recently cloned a library of 80 malarial antigens into a mammalian surface expression vector pDisplay (Invitrogen; Chia et al., 2014). They transfected these expression vectors into mammalian

cells and the antigens were expressed on the cell surface, creating a library of antigen-presenting cells. The library was screened against immune and non-immune sera obtained from mice immunized with live sporozoites or blood parasites under drug cover (Belnoue et al., 2008). Similarly, all immunized volunteers in the study were protected from sporozoites challenge. The antibody repertoire of immunized volunteers was extremely broad and varied. MAEBL was found to be strongly associated with protection. MAEBL has been implicated in invasion into hepatocytes by sporozoites and merozoites into erythrocytes (Kappe et al., 1998, 2001). It was previously demonstrated that anti-MAEBL antibodies to inhibit *P. yoelii*sporozoites invasion *in vitro* in primary mouse hepatocytes, suggesting that MAEBL is a promising attractive vaccine candidate (Singh et al., 2004).

This approach removes the difficulty of expressing and purifying antigens as soluble recombinant proteins. However, the library size in this study represents a small representation of the entire *Plasmodium* genome (∼1%) and antigens screened were mainly restricted to those with reasonably good transfection efficiency.

#### **SCREENING WHOLE PARASITE LYSATES AGAINST AFFINITY-PURIFIED IgG FROM NATURALLY SURVIVING MICE**

Due to ethical and technical restrictions, identification of protective immune correlates in humans can only be evaluated in the peripheral blood component. This hinders the study of a vital stage of the parasite's life cycle, the liver stage. It also limits the examination of the host immune responses in the secondary lymphoid organs such as the spleen, which are also important sites for the induction of protective immunity.

The use of comparative genomic analysis, through the construction of genome-wide synteny maps, has identified other non-human models of malaria (Carlton et al., 2002; Tachibana et al., 2012). Mouse models have played a vital role in the understanding of the immunobiology of malarial infections. The mouse malaria parasite, *P. yoelii*, has been used as a model for malaria research due to the substantial similarity to the human parasite *P. falciparum* (Hall et al., 2002). In addition to being cheaper to maintain and easier to handle, the mouse also has a well characterized immune system, hence offering great advantages over non-human primate models (Wykes and Good, 2009; Taylor-Robinson, 2010). Infection studies of rodent malaria species and their hosts have provided information on parasite biology and pathogenicity. Identification of orthologs allows preclinical validation of new chemotherapies and vaccine candidates. The lethal *P. yoelii* model, which causes death in BALB/c mice, is an excellent model for investigating the vaccine efficacy of the candidates *in vivo* (Langhorne, 1994). Kamali et al. (2012) observed that 20% of ICR mice infected with the lethal strain of *P. yoelii* cleared the infection and these naturally surviving mice had boosted immunity following a second challenge. They affinity-purified the IgG from these mice and probed against whole parasite lysates. Using MALDI-TOF analysis, the antigenic specificities of the protective IgG were Heat shock protein 70 (HSP70), protein disulphide isomerase, plasmepsin and a 39 kDa subunit of eukaryotic translation initiation factor 3. Although the authors did not validate the protective potential of the identified antigens, the approach used in the study identified novel antigens (with the exception of PfHSP70.1 (Haddad et al., 2004). Antibodies against PfHSP70.1 have been shown to eliminate *in vitro* liver stage parasites through antibody-dependent cell-mediated cytotoxic mechanisms (Renia et al., 1990).

#### **SCREENING FOR PROTECTIVE DNA VACCINE CANDIDATE IN MOUSE CHALLENGE MODEL**

New molecular technologies such as Gateway cloning make it easy to clone large numbers of genes into plasmids. Using this technology, Haddad et al. (2004) produced an expression library of 182 *Plasmodium* exons coding for pre-erythrocytic antigens in the mammalian immunization vector VR1012. They immunized mice with 19 out of the 182 cloned vectors, either singly or in combination, and assessed vaccine efficacy following sporozoites challenge by examining the ability of the immunized mice to reduce liver stage parasite load. The most promising DNA vaccine candidate identified was Py01316, annotated as a Qa-SNARE protein in PlasmoDB, which gave a 68–79% reduction in parasite load.

The approach taken by Haddad et al. (2004) has the benefit of generating a library of DNA vaccine candidates, targeting many *Plasmodium* genes with great ease, and does not require the laborious process of generating recombinant proteins. It also provides information on the immunogenicity of the DNA constructs and *in vivo* effectiveness of the induced immune responses to reduce parasite load. However, screening for protective DNA vaccine targets in mice is laborious and requires a large number of mice. As a result, the authors screened a total of 19 out of the 182 cloned plasmids, which limited the number of malarial antigens that can be validated for protective efficacy.

#### **TARGETED SCREENING TO IDENTIFY MALARIAL ANTIGENS AND THEIR RECEPTORS**

Crosnier et al. (2011) expressed a library of erythrocyte surface proteins with the mammalian expression system. Using the avidity-based extracellular interaction screen (AVEXIS), they screened recombinant PfRh5 protein (the bait protein), which is critical for erythrocyte invasion by merozoites, against the library of erythrocyte surface proteins (the prey protein). The PfRh5 protein was found to interact with only one erythrocyte surface protein, basin. The anti-basin monoclonal antibodies were highly effective at blocking *P. falciparum* invasion into erythrocytes *ex vivo* and these blocking effects were efficacious with 15 other culture-adapted and field strains of *P. falciparum*. These data have implications for novel therapeutics. While this does not directly identify new protective malarial antigens for vaccine development, the identification of basin as the binding partner of PfRh5 and perhaps further characterisation studies on the expression levels of basin by the target population would provide critical information on the vaccine efficacy of PfRH5, should PfRh5 be developed as a vaccine candidate. Understanding how basigin binds to PfRh5 could also facilitate rational design of drug compounds to block *P. falciparum*'s invasion into erythrocytes.

One of the shortcomings of AVEXIS is the generation of the library of recombinant proteins, which is laborious. This approach also requires the prior identification of a known bait protein, which is difficult for identifying novel host–parasite interacting antigen pairs. However, the AVEXIS assay is advantageous in its ability to detect direct low-affinity protein interactions, which might not be detectable in other screening methods. More recently, this type of library has shown to be a powerful tool to identify new protective antigens for potential vaccine target development. Using sera from a longitudinal study in a cohort of Kenyan children, Osier et al. (2014) identified 10 antigens (PF3D7\_1136200, MSP2, RhopH3, P41, MSP11, MSP3, PF3D7\_0606800, AMA1, Pf113, and MSRP1) that were associated with protection against clinical episodes of malaria. While the AVEXIS approach has been used to identify blood stage antigens in this study, the study could be extended to include antigens for other stages.

#### **IDENTIFICATION OF PROTECTIVE PRE-ERYTHROCYTIC ANTIGENS**

Pre-erythrocytic antigens have been attractive targets for vaccine development. This is mainly due to the demonstration that sterile immunity against malaria is achievable in experimental sporozoite challenge experiments in humans following vaccination with whole sporozoites (Hoffman et al., 2002; Roestenberg et al., 2009; Seder et al., 2013).

Most of the above described approaches for identification of protective antigens have used sera from chronically exposed individuals living in endemic regions to screen for protective malarial antigens. Although the approaches have identified mainly blood stage antigens, some of the identified blood stage antigens, such as AMA1 (Silvie et al., 2004), TRAP (Robson et al., 1995), EBA175 (Gruner et al., 2001b), PfEMP3 (Gruner et al., 2001a), and HSP70.1 (Renia et al., 1991), which are also expressed during the liver stage.

To screen for protective pre-erythrocytic antigens, it is essential to have the appropriate sera sets against which the library of malarial antigens will be screened. While sera from chronically exposed individuals living in endemic regions are relatively easier to obtain, they are more likely to have an antibody repertoire that is predominantly specific for blood stage antigens, hence might not be suitable for the identification of liver stage antigens. Marchand and Druilhe (1990) and Gruner et al. (2003) were the first to show in human that chloroquine prophylaxis protected against blood stage infection and use the protected sera to screen for protective antigens. This led to the identification of liver-specific (LSA1; Guerin-Marchand et al., 1987) or cross stagespecific antigens stages (LSA-3, STARP, and SALSA; Fidock et al., 1994; Bottius et al., 1996; Daubersies et al., 2000). Recently, the studies by Trieu et al. (2011) and Chia et al. (2014) involved the use of sera from animals immunized with sporozoites. Trieu et al. (2011) identified 16 previously uncharacterized pre-erythrocytic antigens (Trieu et al., 2011), while the pre-erythrocytic MAEBL antigen was identified by Chia et al. (2014).

#### **CONCLUSION**

Historically, vaccine development efforts have been focused on immunodominant antigens as vaccine candidates such as merozoite or sporozoite surface proteins. However, the success has been limited thus far.

More recently, through the use of new technologies, immunoscreens have become more comprehensive, and has revealed the strong association of non-immunodominant malarial antigens with protection. Antigen libraries, expressed in bacterial, mammalian, or wheat germ cell-free expression systems, are created either as DNA libraries, recombinant proteins or on cell surface as antigen-presenting cells. In the screening of these antigen libraries for protective antigens, immune sera are an important tool. The type of immune sera chosen for the screening is critical. Carefully planned vaccination trials with an experimental challenge provide differential groups of sera from protected versus non-protected vaccinated individuals to identify protective antigens. Sera from chronically exposed individuals living in malaria-endemic regions have been also used in studies to screen for protective antigens. While sera from these individuals inform about naturally acquired immunity, it is essential to differentiate between the susceptible and the resistant individuals in these naturally exposed individuals. These differential sera allow the exclusion of immunodominant antigens, and inclusion of the non-immunodominant antigens that are associated with protection.

Taken together, the current consensus is protection against malaria is attributed to robust humoral responses directed against a panel of various non-variant antigens instead of only a single or a few immunodominant antigens. Studies to validate the feasibility of these minor non-immunodominant antigens as vaccine candidates should be prioritized for vaccine development against malarial infections. In addition, understanding antigen recognition is an essential step for the establishment of key immune correlates of protection against malarial infections, which would aid greatly in validating vaccine efficacy.

#### **ACKNOWLEDGMENTS**

This work was supported by an intramural grant from Singapore's Agency for Science, Technology and Research. Wan Ni Chia is supported by a postgraduate scholarship from the Yong Loo Lin School of Medicine, National University of Singapore (Singapore).

#### **REFERENCES**

surface antigen of *Plasmodium falciparum* are associated with age in a malaria endemic area. *Mol. Cell. Proteomics* 10:M111. doi: 10.1074/mcp.M111.008326

as targets of protective human immunity to *Plasmodium falciparum* malaria for vaccine and biomarker development. *J. Immunol.* 191, 795–809. doi: 10.4049/jimmunol.1300778

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 19 August 2014; accepted: 17 October 2014; published online: 17 November 2014.*

*Citation: Chia WN, Goh YS and Rénia L (2014) Novel approaches to identify protective malaria vaccine candidates. Front. Microbiol. 5:586. doi: 10.3389/fmicb.2014.00586 This article was submitted to Microbial Immunology, a section of the journal Frontiers in Microbiology.*

*Copyright © 2014 Chia, Goh and Rénia. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## APC licensing and CD4 T cell help in liver-stage malaria **+**

#### *Ian N. Crispe\**

Department of Pathology, University of Washington, Seattle, WA, USA

#### *Edited by:*

Ute Frevert, New York University School of Medicine, USA

#### *Reviewed by:*

Muriel Moser, Université Libre de Bruxelles, Belgium Laurel L. Lenz, University of Colorado School of Medicine, USA Xun Suo, China Agricultural University, China

#### *\*Correspondence:*

Ian N. Crispe, Department of Pathology, University of Washington, 1959 North East Pacific Street, Seattle, WA 98195, USA e-mail<EMAIL_ADDRESS>
Malaria parasites spend a critical phase of their life cycle inside hepatocytes, in an environment with complex and distinctive immunological features. Here I will discuss how the immunological features of the liver and the adaptations of malaria parasites interact, resulting in defective CD8+ T cell immunity. These processes are explored with a focus on the mechanism by which CD4+ T cells deliver help to CD8+ T cells, and specifically through their interaction with antigen-presenting cells (APCs), resulting in "licensing" of the APCs and enhanced capacity to optimally activate CD8+T cells. Synthesis of the available evidence supports a model in which the parasite-mediated manipulation of programmed cell death in infected hepatocytes impairs the capacity of the liver's immune system to successfully license APCs and fully activate T cell immunity.

**Keywords: liver, hepatocyte, malaria parasite, immunity, CD4+ T cell help, CD8+ T cell, antigen-presenting cells, licensing**

#### **INTRODUCTION**

Malaria parasites are vulnerable to the mammalian immune system from the time they penetrate the skin as sporozoites, during the passage of these pre-hepatic forms through the blood to the liver, during their residence inside hepatocytes, and after their emergence, when they parasitize erythrocytes. In this review, I consider only immunity to the hepatocellular stage of the parasite. Liver-stage malaria parasites gain access to hepatocytes by crossing the liver sinusoidal endothelium, and both direct and indirect evidence suggests that they may exploit Kupffer cells, the liver's large resident macrophage population, as portals to gain access to the underlying Space of Disse, and thus to hepatocytes (Frevert et al., 2005; Baer et al., 2007).

Once inside the hepatocyte, the malaria parasite establishes a vacuole, the membrane of which contains both host-derived proteins, and proteins secreted by the parasite. This parasitophorous vacuole separates the parasite from the host cell cytoplasm, and acts as the interface through which the parasite subverts cellular processes to protect it from host defense and to provide a supply of nutrients. The parasite's interactions are highly adapted and successful. Thus, young children infected for the first time with malaria show little resistance, but over time and with repeated antigen exposure, immunity to the blood-stage gradually established sufficient control over parasite growth that the severity of clinical illness is reduced. However, it is not clear that repeated infection engenders any immunity against the liver-stage itself. Therefore, to study immunity to the liver-stage requires the use of animal models.

While natural immunity to the liver-stage is difficult to detect, artificial immunization protocols clearly generate such immunity, manifest both as reduced parasite load in the liver and as a delay, or the prevention of blood-stage infection. One approach is to infect a murine or a human subject with radiation-attenuated parasites (Hoffman et al., 2002). This approach results in sterilizing immunity in both species, and in mouse models a variety

of host defenses have been implicated, including innate and antigen-specific immune cells (NK cells, CD4+ T cells, CD8+ T cells) and cytokines (IL-12, IFN-gamma; Doolan and Hoffman, 2000). The development of techniques to modify *Plasmodium spp.* genes has led to an alternative approach: the development of genetically attenuated parasites (GAPs). While human trials are only beginning (Spring et al., 2013), GAP can produce solid protection in mouse challenge models (Jobe et al., 2007; Trimnell et al., 2009; Vaughan et al., 2010).  In principle, a CD8+ T cell can manifest either or both of these effector functions *in vivo*; for example in viral hepatitis B, the major anti-viral effect appears to be due to IFN-gamma secretion, rather than cytotoxic killing (reviewed in Guidotti and Chisari, 2001). However, a recent *in vivo* study shows that GAP immunization, followed by boosting with a DNA vaccine, results in host defense that can destroy *in situ* hepatocytes that express malaria-encoded antigens (Chen et al., 2014). These data support the working hypothesis that the central effector mechanism against liver-stage malaria parasites is cytotoxic killing of the infected cells by CD8+ cytotoxic T lymphocytes (CTLs), rather than cytokine-driven intracellular cure of the infection.

#### **THE IMPORTANCE OF HELP**

The fundamental immunological problem in liver-stage malaria is to account for the failure of effective immunity during natural malaria infection, but the capacity of irradiated sporozoites and GAP to induce such immunity. The role of CD4+ T cells in such immunity is complex. Direct antibody-mediated depletion of CD4+ T cells at the time of challenge does not impair anti-malaria immunity to the liver-stage, implying that at the effector level, CD8+ CTL are sufficient (Tarun et al., 2007). Yet experiments in animals that congenitally lack CD4+ T cells are consistent with the concept that CD4+ T cells are required to prime effective

immunity (Overstreet et al., 2011). In terms of the biology of CTL maturation and memory, this makes complete sense. In host defense against intracellular bacteria, in anti-viral immunity, and particularly in CD8+ T cell responses against non-inflammatory antigens such as a minor histocompatibility antigens, CD4+ T cell help is essential, but the nature of the requirementfor help depends on the nature of the antigen. Thus, minor histocompatibility antigens require CD4+ T cells to induce an effective CD8+ T cell response, while other antigens need CD4+ T cells to ensure either the long-term survival, or the memory effector function of CD8+ T cells (Prlic et al., 2007). There is a rough inverse correlation between the extent to which the antigen is accompanied by accessory signals, including pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), and the extent to which CD4+ T cell help is essential for a CD8+ T cell response. In viral hepatitis C, the main cause of immune failure and chronic infection appears to be the lack of a CD4+ T cell response, resulting in CD8+ T cells that become exhausted over time (Thimme et al., 2001; Grakoui et al., 2003; Radziewicz et al., 2007; Mueller et al., 2010). This same exhausted phenotype in CD8+ T cells is a major feature of immune failure in many potentially immunogenic cancers (Pardoll, 2012). These diverse diseases illustrate the central importance of CD4+ T cells in most CD8+ CTL responses; but their precise role in immunity to liver-stage malaria has yet to be defined.

As a complex parasite, *Plasmodium spp.* encode diverse nonmammalian proteins and synthesize many other non-mammalian molecules that could serve as PAMPs, but during the liver-stage these molecules could be sequestered inside the parasitophorous vacuole. However, this is clearly not the case for all CTL target antigens, since in the context of immunity induced by either irradiated sporozoites or GAP, infected hepatocytes can be killed. There is evidence that *Plasmodium spp.* may actively manage DAMPs, particularly those associated with cell death. Thus, infection of mouse hepatocytes with *Plasmodium yoelii* results in decreased p53, a mediator of G1 cell cycle checkpoint arrest, and a drug that sustained p53 expression reduced infection (Kaushansky et al., 2013a). The death of hepatocytes infected with wild-type (WT) parasites was driven by mitochondria and impaired by Bcl-2, while such control of host cell death appeared to be linked to the presence of a parasitophorous vacuole (Kaushansky et al., 2013b). Thus it seems that the parasite first delays the division of infected stressed hepatocytes, then inhibits their death, and finally allows them to die via the mitochondrial pathway of apoptosis. All of these maneuvers may sequester both DAMPS and PAMPs, eliminating pathways of immune activation.

Together with accessory signals from PAMPs and DAMPs, maturation of an effective CTL response with protective memory depends on CD4+ T cell help. In the case of malaria liverstage antigens, the parasites reside in a cell type that expresses major histocompatibility complex (MHC) class I, but not MHC class II. Since CD4+ helper T cells are activated by antigenpresenting cells (APCs) that express MHC class II, it is clear that for CD4+ T helper cells to develop, malaria-encoded antigens must be presented by a different cell type, a mechanism termed cross-presentation; the T cell activation that follows is cross-priming (Bevan, 2006). Cross-presentation may occur by the uptake of cellular fragments or soluble proteins, or by the transfer of complete MHC-antigen complexes (termed: cross-dressing; Wakim and Bevan, 2011). In the liver, many cell types have the potential to take up antigen and engage in cross-presentation. Thus, there are liver-resident and circulating dendritic cells (DCs), Kupffer cells, liver sinusoidal endothelial cells (LSECs) and potentially also hepatic stellate cells (Ebrahimkhani et al., 2011). Among these, liver DCs and LSECs have the strongest credentials (Berg et al., 2006; Sumpter et al., 2007). Caveats concerning their role as APCs for liver-stage malaria antigens will be considered below.

#### **LICENSED VERSUS UNLICENSED HELP**

Once a CD4+ T helper cell is activated, help may be delivered to a CD8+ T cell by several known mechanisms (Lee et al., 2003). Licensing of theAPC occurs when the CD4+T cell recognizes antigen presented by an MHC class II+ APC, and delivers activating signals through co-stimulatory pathways, involving such receptors as CD80, CD86, and CD40 on the APC and their counter-receptors on the T cell. These signals activate the APC, resulting it its capacity to deliver enhanced signals to any CD8+ T cell that recognizes antigen on the same APC (Smith et al., 2004). This mechanism, though elegant and precise, requires that two relatively rare cells, the antigen-specific CD4+ T cell and the antigen-specific CD8+ T cell, should find the same APC, but it does not require that they should do so at the same time. While recent advances in *in vivo* imaging clearly reveal sustained contact between both CD4+ T cells and CD8+ T cells with DCs, clear evidence for simultaneous clusters of all three cells is not a striking feature of active immune responses in lymph nodes (Germain et al., 2012). Licensing also clearly requires that the APC express MHC class I and MHC class II, while hepatocytes do not. Abundant evidence suggests that hepatocyte can act as APC and induce primary activation in naïve CD8+ T cells, both *in vitro* and *in vivo* (Bertolino et al., 1998; Klein and Crispe, 2006), but the lack of MHC class II expression means that a hepatocyte is *an APC that cannot be licensed*, even in principle. Therefore if anti-malaria CD8+ T cells were to be primed directly on hepatocytes, they could only receive help through an alternative mechanism.

Two known possibilities exist to bypass the need for licensing. First, and most simply, the CD4+ T cell may secrete growth factors such as Interleukin-2 (IL-2) that promote CD8+ T cell proliferation and maturation. This model is supported by experiments in which CD8+ T cells, selectively deprived of the ability to respond to IL-2, fail to undergo full differentiation (Williams et al., 2006). The alternative mechanism through which CD4+ T cells can deliver help to CD8+ T cells is through a direct interaction that depends on CD40 on the CD8+ T cell, and appears to be a direct T–T interaction involving on CD40L on the CD4+ T cell (Isogawa et al., 2013). This mechanism of help allows for the CD8+ and the CD4+ T cell to interact with different APCs, and so could overcome the limitations of a CTL target antigen that is not readily cross-presented.

To understand how fully effective, long-lived CD8+ T cells might arise in response to *Plasmodium*-infected hepatocytes, it is important to know whether these CD8+ T cells are primed directly on hepatocytes, or via cross-presentation of antigen

may exit from the parasitophorous vacuole (PV ) in some kind of transport vesicle (ves), and be directly presented to CD8+ T cells by the infected hepatocyte. 2: Hepatocyte antigens may be taken up and cross-presented by neighboring hepatocytes, resulting in cross-presentation to CD8+ T cells. However, hepatocytes do not express MHC class II, and so cannot be licensed. This presents an obstacle to the delivery of CD4+ T cell help to these CD8+ T cells. 3: Malaria liver-stage antigens may be cross-presented

by an APC that can be licensed. There is some circumstantial evidence that cross-presentation of hepatocellular antigens is constrained. First, in experimental delivery of antigen to hepatocytes using an adeno-associated virus (AAV) vector, the activation of antigen-specific CD8+ T cells was exclusively via non-bone marrow-derived APC, consistent with direct-priming (Wuensch et al., 2010). Second, in a liver transplant patient infected with Hepatitis C, new CTL appeared that were restricted to the human leukocyte antigen (HLA) of the liver donor, implying that they were primed on a solid tissue and not on bone marrow-derived DCs (Lauer, 2005). Third, when hepatocytes undergo apoptotic death, a major pathway of their disposal is phagocytosis by other hepatocytes (Dini et al., 1992; Dalton et al., 2003), which would keep any pathogen-encoded antigens within the hepatocyte pool. These considerations render it plausible that hepatocellular antigens are limited in their susceptibility to cross-presentation.

However, these experiments are subject to the caveat that LSECs are non-bone marrow-derived APCs that are not replaced with host-derived cells after a liver transplant. They are efficient in antigen uptake, and fully competent to cross-present both hepatocyte antigens and cancer cell-derived antigens *in vitro* (Berg et al., 2006; Ebrahimkhani et al., 2011). Furthermore these cells express both MHC class I and MHC class II, and so could potentially be

both MHC class I and class II, it can be licensed, facilitating CD4+ T cell help for the CD8+ T cell. 4: At the time of death of the infected hepatocyte and merozoite release, antigens may be cross-presented by myeloid cells, including liver-resident Kupffer cells (KC) and recirculating dendritic cells (DC). These cells also express MHC class I and class II, and are optimized to transmit CD4+ T cell help for CD8+ T cells through the licensing mechanism.

licensed. *In vitro*, the activation of CD8+ T cells by LSECs typically leads to tolerance, but this could be in their un-licensed state; antigen recognition by a CD4+ T cell on an LSEC could result in licensing, with a changed outcome and complete activation when a CD8+ T cell interacts with the same LSEC. Kupffer cells occupy a rather similar position, since they are MHC class I+ II+ macrophages, and a subset of them termed sessile Kupffer cells are both radiation-resistant, and persistent after a liver transplant (Kennedy and Abkowitz, 1997; Klein et al., 2007). *In vitro*, Kupffer cell also induce abortive T cell activation leading to tolerance, but explicit experiments to license these cells via CD4+ T cell help, and then test their capacity to prime CD8+ T cells, have not been attempted.

#### **THE ROOTS OF HELPLESSNESS**

The mechanisms of direct versus cross-presentation, CD4+ T cell priming and the delivery of help via licensed APCs or otherwise collectively define the problem with respect to liver-stage malaria vaccine strategies (**Figure 1**). Any of the constraints that limit effective delivery of CD4+ T cell help to CD8+ T cells could apply in the context of natural malaria infection, but be circumvented by irradiated sporozoites or GAP vaccines.

First, viable malaria parasites could facilitate the death of the infected hepatocyte by a mechanism that favors the containment of PAMPs and DAMPs. Either irradiated sporozoites, or GAP that act as live-attenuated vaccines, may lose this ability. This would have the effect of rendering malaria antigen-specific CD8+ T cells highly help-dependent due to the limited PAMP and DAMP signals. Second, the hepatocytes that are infected by wild-type parasites could undergo death in a manner that facilitates uptake by neighboring hepatocytes. Hepatocytes are surprisingly phagocytic. They can take up activated CD8+ T cells and destroy them in a mechanism of tolerance termed suicidal emperipolesis (Benseler et al., 2011); and they can take up apoptotic hepatocytes through a mechanism that depends on the asialo-glycoprotein receptor (McVicker et al., 2002; Dalton et al., 2003). Such uptake would tend to contain parasite-encoded antigens within an APC that can engage CD8+ T cells, but cannot be licensed, resulting in impaired susceptibility to CD4+ T cell help. It is then possible that irradiated sporozoites or GAP result in hepatocyte death from a different mechanism, such as pyroptosis or necroptosis, that facilitates cross-presentation by other cell types. So far these issues have been considered in cancer, where many anti-cancer drugs kill cancer cells by diverse mechanism that may influence antigen presentation (Guo et al., 2014); but the principle is likely to apply also to infectious disease, in particular malaria parasites in hepatocytes.

In addition to regulation at the levels of antigen recognition and APC licensing, T cell immune responses, including the responses to many hepatocellular pathogens, are regulated through multiple classes of inhibitors molecules, including coinhibitory signals such as PD-L1 and Galectin-9 that interact with counter-receptors (PD-1, and Tim3 respectively) on the T cells; and immune suppressive small molecules such as kynurenine, the product of tryptophan breakdown by the enzyme indoleamine 2,3-dioxygenase (IDO), and prostaglandin-E2, which is made in immunosuppressive macrophages by an enzyme cascade involving cyclo-oxygenase type -2 (COX2; reviewed in Crispe, 2014). Several of these mechanisms have already been implicated in resistance to blood-stage malaria infection (Butler et al., 2012), but their significance in regulating immunity to the liver-stage had not been documented.

Such co-inhibitory signals should not be seen as an alternative explanation to the limitations placed on CD4+ T help by constraints of cross-presentation and APC licensing. One striking example comes from virus hepatitis research, where the "exhausted" state of anti-hepatitis B virus (HBV) CD8+ T cells could be reversed by providing CD40, a key component of the licensing mechanism (Isogawa et al., 2013). Typically, CD8+ T cells in chronic HBV and HCV display all the features of exhaustion, and in both cases there is a lack of CD4+ T cell activation. An inclusive view is that the display of PD-1, Tim3, and Lag3 on the exhausted CD8+ T cell may be a downstream consequence of the inability to deliver CD4+ T cell help. In malaria, as in other globally important liver pathogens, it will be most important to understand whether this results from a failure of APC licensing, or the failure of those mechanisms that have evolved as back-ups in case licensing fails.

#### **CONCLUSION**

Liver-stage malaria parasites inside hepatocytes occupy a cell type that can actively present antigen to MHC class I-restricted CD8+

T cells, but can neither directly activate CD4+ T cells, nor become licensed through interaction with CD4+ T cells. Since CD4+ T cell help is essential for full CD8+ T cell activation, function and protective memory, it is critical that both non-specific danger signals (DAMPs) and pathogen-associated innate immune activators (PAMPs) converge on an APC that can both cross-present antigen to CD4+ T cells, and be licensed to fully activate CD8+ T cells. In WT malaria infection, the parasite deftly subverts these mechanisms, in part through exerting control over the timing and mode of death of the infected cell. Successful experimental malaria vaccine strategies involve liver-attenuated parasites that furnish antigens, DAMPS and PAMPs, but fail to exert full control over the death of the infected cell. More detailed understanding of these mechanisms may lead to techniques to optimally deliver practical subunit vaccines, bringing CD4+ T cell help to anti-malaria CD8+ T cells and resulting in long-lived protective immunity at the liver-stage.

#### **REFERENCES**

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 26 August 2014; paper pending published: 19 October 2014; accepted: 29 October 2014; published online: 11 November 2014.*

*Citation: Crispe IN (2014) APC licensing and CD4*+ *T cell help in liver-stage malaria. Front. Microbiol. 5:617. doi: 10.3389/fmicb.2014.00617*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Microbiology.*

*Copyright © 2014 Crispe. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

**MINI REVIEW ARTICLE** published: 18 November 2014 doi: 10.3389/fmicb.2014.00625

### Antibody and B cell responses to Plasmodium sporozoites

#### *Johanna N. Dups1, Marion Pepper 2 and Ian A. Cockburn1\**

1 Department of Pathogens and Immunity, John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia

2 Department of Immunology, School of Medicine, University of Washington, Seattle, WA, USA

#### *Edited by:*

Urszula Krzych, Walter Reed Army Institute of Research, USA

#### *Reviewed by:*

Xun Suo, China Agricultural University, China Hajime Hisaeda, Gunma University, Japan Peter Dobbs Crompton, National Institutes of Health, USA

#### *\*Correspondence:*

Ian A. Cockburn, Department of Pathogens and Immunity, John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia e-mail<EMAIL_ADDRESS>
Antibodies are capable of blocking infection of the liver by Plasmodium sporozoites. Accordingly the induction of anti-sporozoite antibodies is a major aim of various vaccine approaches to malaria. In recent years our knowledge of the specificity and quantities of antibodies required for protection has been greatly expanded by clinical trials of various whole sporozoite and subunit vaccines. Moreover, the development of humanized mouse models and transgenic parasites have also aided our ability to assess the specificity of antibodies and their ability to block infection. Nonetheless, considerable gaps remain in our knowledge – in particular in understanding what antigens are recognized by infection blocking antibodies and in knowing how we can induce robust, long-lived antibody responses. Maintaining high levels of circulating antibodies is likely to be of primary importance, as antibodies must block infection in the short time it takes for sporozoites to reach the liver from the skin. It is clear that a better understanding of the development of protective B cell-mediated immunity will aid the development and refinement of malaria vaccines.

**Keywords: malaria,** *Plasmodium***, B cells, antibodies, sporozoites, pre-erythrocytic stages**

#### **INTRODUCTION**

The generation of protective antibodies underpins the success of almost all of our current vaccines (Plotkin, 2008, 2010). While there is no licensed vaccine available for malaria, one indication that a vaccine may be achievable comes from the seminal finding that immunization with radiation attenuated sporozoites (RAS) results in protection against live parasite challenge in both mice and humans (Nussenzweig et al., 1967; Clyde et al., 1973; Hoffman et al., 2002; Seder et al., 2013). Moreover, complete protection against malaria has also been demonstrated in volunteers immunized with low numbers of infectious bites under chloroquine prophylaxis (Roestenberg et al., 2009), also known as DAP immunization. Protective responses induced by RAS appear to be principally based on CD8+ T cells and antibodies (Schofield et al., 1987; Weiss et al., 1988), with contributions from CD4+ T cells, gamma-delta T cells and natural killer (NK) cells also proposed (Tsuji et al., 1994; Doolan and Hoffman, 1999; Oliveira et al., 2008). Antibodies could block infection at the pre-erythrocytic stages in several ways, either by neutralizing sporozoites directly, opsonizing sporozoites for phagocytosis or blocking invasion of parasites into hepatocytes. There is also some evidence that antibodies can block liver stage development, though the mechanism for this is unclear (Chatterjee et al., 1996). In this minireview we will examine the evidence that antibodies can play important roles in protection, evaluate the targets of those antibodies and determine what needs to be known to advance our knowledge of antibody and B cell immunity to sporozoites, and potentially contribute to vaccine development.

#### **ANTIBODY MEDIATED PROTECTION AGAINST SPOROZOITE CHALLENGE**

#### **EARLY STUDIES ON HUMORAL IMMUNITY: THE IDENTIFICATION OF THE CIRCUMSPOROZOITE PROTEIN AS A TARGET OF PROTECTIVE IMMUNITY**

Shortly after the discovery of protective immunization with RAS, it was found that RAS immunized mice rapidly cleared the sporozoite inoculum from circulation, suggesting the presence of neutralizing antibodies (Nussenzweig et al., 1972). Later transfer experiments showed that immunoglobulin G (IgG) and T cells acted synergistically to confer sterile immunity to sporozoites, (Schofield et al., 1987). More recently, sera from individuals immunized with DAPs was found to reduce infection of humanized mice infected with *Plasmodium falciparum*(Behet et al.,2014). Other early evidence for a role of antibodies in protection came from the identification of mAbs capable of inducing the precipitation of material from the surface of human and rodent malaria sporozoites – a phenomenon known as the circumsporozoite reaction (Yoshida et al., 1980). These mAbs were shown to be capable of blocking infection *in vitro* (Nardin et al., 1982), and *in vivo* (Potocnjak et al., 1980). Subsequently the target of these antibodies was cloned and identified as the CSP (Ellis et al., 1983; Dame et al., 1984; Enea et al., 1984)

CSP is a GPI-anchored protein consisting of a conserved domain structure with N- and C-terminal domains separated by an asparagine-rich repeat region. The C-terminal domain contains a conserved TSR, which is important for the recognition and binding of hepatocytes (Cerami et al., 1992; Frevert et al., 1993). The N-terminal domain acts by masking the TSR of the C-terminal domain, and has to be cleaved to allow the parasite to invade hepatocytes (Coppi et al., 2011). In contrast, the role of the repeat region, which in *P. falciparum* consists of (NANP)n

**Abbreviations:**AMA-1, apical membrane antigen 1; CSP, circumsporozoite protein; DAP, drug arrested parasites; LLPC, long-lived plasma cell; LSA-1, liver stage antigen 1; mAb, monoclonal antibody; STARP, serine threonine and asparagine rich protein; TRAP, thrombospondin repeat anonymous protein; TSR, thrombospondin repeat.

repeats with a few NVDP repeats interspersed at the beginning, is unknown. Nonetheless, this region was identified early on as an important target of protective immunity, and contains the epitopes recognized by all the original anti-CSP mAbs reported (Zavala et al., 1983, 1985). In terms of protective immunity, much less work has been done to investigate antibody responses to the N- and C-terminal domains despite their functional importance. Several studies have shown that immunization with N-terminal peptides can induce invasion-blocking antibodies (Rathore et al., 2005; Bongfen et al., 2009). Interestingly, a correlation between the presence of antibodies to this region with a reduction in malaria morbidity has also been observed (Bongfen et al., 2009).

#### **PROTECTION MEDIATED BY ANTI-CSP ANTIBODIES IN HUMANS: LESSONS FROM VACCINE TRIALS**

Perhaps, the strongest evidence that anti-CSP antibodies can be protective comes from trials of the CSP-based RTS,S vaccine (Stoute et al., 1997). RTS,S is a virus-like particle consisting of 19 NANP repeats and C terminal domain of the CSP fused to the Hepatitis B Surface antigen. RTS,S is currently in Phase III clinical trials in a formulation with AS01, a proprietary adjuvant consisting of a mixture of liposomes, monophosphoril lipid A and saponin (Casares et al., 2010). In experimental challenges of malaria-naïve adults, RTS,S gives short-lived sterile protection in around 50% of volunteers (Kester et al., 2001, 2009). In phase III clinical trials in endemic areas, RTS,S gave 56% protection against clinical malaria among 5–17 month old children (Agnandji et al., 2011b), and 31% efficacy among younger infants (Rts et al., 2012). While there is some evidence of reduced numbers of infections in the field (Guinovart et al., 2009), the main effect of the vaccine appears to be on disease severity, which is surprising as CSP is not expressed in the pathogenic blood stages. The data are however similar to the findings of Bongfen et al. (2009; described above) showing protection against disease correlating with high titres of N-terminus specific antibodies. One explanation for these results is that the vaccine might lower the initial inoculum of parasites and thus the number of blood stages emerging from the liver, buying time for the immune system to control infection. It may also be that while the vaccine does not block all infectious bites, the breakthrough infections are less likely to be genetically complex or highly virulent (Moorthy and Ballou, 2009). Importantly, these data rebut one of the traditional objections to pre-erythrocytic stage vaccines, namely that they would be ineffective if parasites do breakthrough and establish blood stage infection. Nonetheless, this does not negate the importance of developing vaccines targeting other life cycle stages in tandem with pre-erythrocytic approaches.

The mechanisms of protection by RTS,S are poorly understood, with different trials measuring different immunological parameters. Most studies report ELISA titres of total IgG responses against the (NANP)n repeat, but some use μg/ml while other report titres as ELISA Units (EU). Only in early studies were antibodies segregated by subclass, with no association reported between subclasses and protection (Stoute et al., 1997; Kester et al., 2001). Antibodies to the C-terminal domain have been little studied and were not found to be associated with protection (Kester et al., 2001). Finally

only one study reports the number of CSP-specific B cells in vaccinated individuals (Agnandji et al., 2011a). Nonetheless direct evidence that RTS,S induced antibodies can protect comes from a recent study in which human mAbs targeting the CSP repeat were shown to block *P. falciparum* infection of humanized mice (Foquet et al., 2014).

Clinical trials of the vaccine both in naïve individuals and in the field also provide strong evidence of a role for anti-CSP antibodies. Mathematical modeling of a Phase IIb trial in which malaria naïve volunteers were given the RTS,S vaccine formulated in either AS01B or AS02A (Kester et al., 2009), suggested that the bulk of protection comes from high levels (100–200 μg/ml) of anti-repeat antibodies, aided by robust CD4+ T cell responses (White et al., 2013). Importantly, in this study protection did not correlate with titres of Hepatitis B antibodies – which are also induced by the vaccine – suggesting that the CSP antibodies were mediating protection and were not merely a correlate of vaccine "take" (Kester et al., 2009). In endemic areas, mathematical modeling based on a meta-analysis of all Phase II trials has suggested a threshold for infection blocking immunity around 51 EU/ml among children and infants, which is probably similar to the levels required for protection in naïve volunteers (White et al., 2014).

#### **ANTI-CSP ANTIBODIES: OUTSTANDING QUESTIONS**

Regardless of the exact measurement used, it is clear that very high titres of antibody are required for protection. While it is difficult to compare directly with other vaccine regimens, the protective cutoff for vaccines to *Haemophilus influenzae* and *Pneumococcus* are <1 μg/ml (Plotkin, 2008), which leads us to ask, why so much antibody is required for protection against sporozoites? One possible answer is that a high amount of antibody is required to eliminate every sporozoite in the short time it takes for the parasites to exit the skin and migrate to the liver. Nonetheless it is not clear how many of the antibodies measured by ELISA are functional: it may be that only the highest affinity antibodies are capable of sporozoite neutralization. Interestingly, in trials of RAS or DAP, *in vivo* protection and *in vitro* infection blocking is obtained at quite low anti-CSP titres (Seder et al., 2013; Behet et al., 2014; Finney et al., 2014). This may be because these whole parasite vaccines stimulate a broad range of protective immune responses, but it is also possible that they induce antibodies that are better able to recognize the native conformation of CSP. Finally there has been no biophysical or structural characterization of the binding of anti-CSP antibodies. These data would give an idea of the necessary affinity required for sporozoite neutralization and show how antibodies bind to the repeat region, which is likely to be somewhat disorganized (Plassmeyer et al., 2009) – it seems likely that these antibodies may have to stabilize the CSP structure and thus pay a high "entropic cost" in binding.

#### **THE ROLE OF OTHER ANTIGENS IN PROTECTION**

The limited success of CSP-based recombinant vaccines relative to whole parasite approaches has led to a search for other targets of anti-sporozoite antibodies. Immunity to CSP is probably not absolutely required for protection in rodent models as shown by experiments in which mice immunized with wild type *P. berghei* RAS were fully protected against challenge with *P. berghei* sporozoites expressing CSP from either *P. yoelii* or *P. falciparum* (Gruner et al., 2007; Mauduit et al., 2009). Nonetheless, it is unclear how much of this CSP-independent protection is mediated by antibodies rather than T cells.

A variety of potential protective antigens have been identified in the sporozoite and liver stages by classical approaches (Duffy et al., 2012). However, while T cell responses to many of these antigens such as LSA-1 and TRAP are relatively well studied (Duffy et al., 2012; Offeddu et al., 2012), the evidence that antibodies to these proteins may be protective is limited (**Table 1**). Combined antibody titres to CSP, LSA-1 and TRAP have been correlated with reduced incidence of clinical malaria among Kenyan children (John et al., 2005), while antibodies to LSA-1 alone have been associated with protection from reinfection following treatment (Domarle et al., 1999). Early reports suggested that antibodies targeting STARP could have even stronger invasion blocking activity than anti-CSP antibodies, there has been little further examination of this molecule (Fidock et al., 1997). Nonetheless when TRAP and LSA-1 were formulated as vaccines in combination with the AS01 or AS02 adjuvant, they were unable to elicit protection (Cummings et al., 2010; Kester et al., 2014).

More recent studies have used protein microarrays to examine the diversity of antibody responses induced after RAS or DAP immunization of humans (Trieu et al., 2011; Felgner et al., 2013). Antibody profiling of RAS vaccines by protein microarray revealed strong antibody responses to two other established vaccine candidates (AMA1 and TRAP) in addition to CSP and a large number of proteins that had not previously been associated with protection. Many of the remaining proteins were hypothetical or involved in cell cycle functions. Protein microarray analysis of DAP immunized volunteers revealed a different antibody profile with CSP and LSA-1 being the only antigens recognized by all protected individuals. Overall, these studies suggest that no single molecule is the key to protection, rather protective antibody responses consist of broad responses to numerous antigens.

#### **B CELL RESPONSES TO** *Plasmodium* **SPOROZOITES**

While vaccination studies in particular have provided insight into protective antibody responses to sporozoites, sporozoite-specific B cell memory and plasma cell formation is poorly understood. Recognition of a foreign antigen by a B cell receptor leads to the proliferation and differentiation of the activated B cell resulting in the formation of short-lived plasma blasts, "early memory" B cells (that do not enter germinal centers) and germinal center B cells (Zotos and Tarlinton, 2012). Germinal center B cells are generally considered the precursors of LLPCs that can maintain antibody titres (**Figure 1**). Knowledge of the development and maintenance of LLPCs would be of particular interest as the rapid transit of sporozoites from skin to liver offers little or no opportunity for anamnestic responses (memory B cells) to contribute to protection. To the best of our knowledge, no work has been

enter germinal centers where theirB cell receptors undergo somatic hypermutation and affinity maturation. The germinal center B cells are believed to be the major precursors for long-lived plasma cells, which maintain the circulating antibody pool. Memory cells may be class switched or they may retain the IgM+ B cell receptor. The relative contributions of these different memory populations to long term protection against malaria remains an area for further investigation. Figure is based on Taylor et al. (2012). Used with permission from Elsevier.

#### **Table 1 | Potential non-CSP targets of anti-sporozoite antibodies.**

performed in animal models to examine the development and longevity of sporozoite specific B cells and plasma cells. There are however a number of observations of sporozoite specific B cells in naturally exposed and DAP or RTS,S vaccinated individuals. A study in Thailand reported very low levels of B cell responses following natural exposure with only 1/33 adults having CSPspecific B cells (Wipasa et al., 2010). More robust responses are seen following immunization: CSP specific cells accounted for 1% of circulating IgG secreting B cells following RTS,S vaccination and 0.25% of circulating IgG secreting B cells after DAP treatment (Agnandji et al., 2011a; Nahrendorf et al., 2014). These studies are limited however, in that they rely on restimulation enzyme-linked immunospot (ELISPOTs) and therefore cannot provide information on cell phenotype, they report over relatively short timescales and they investigate only IgG antibody secreting cells. Many of the shortcomings of the ELISPOT approach could be overcome by the use of fluorescently labeled antigens to detect rare B cell populations by flow cytometry, however while this has been performed previously for blood stage antigens (Muellenbeck et al., 2013), no such studies have yet been performed with sporozoite antigens.

#### **THE ACTIVATION OF SPOROZOITE SPECIFIC MEMORY B CELLS**

A key outstanding question is how are B cells primed by sporozoites? This is particularly important given that immunization with RAS and DAP represent our most successful immunization approaches. It has been shown that after mosquito biting, a large proportion of parasites remains in the skin and a subset of these migrate to the draining lymph node (Amino et al., 2006). The skin draining lymph node appears to be an important location for the induction of protective immunity to sporozoites: not only is it the first location where sporozoite specific CD8+ T cells are detected, it has also been shown that the removal of this lymph node along with the spleen completely abrogates RAS-mediated protection (Chakravarty et al., 2007). By extension it seems likely that the first interactions of B cells with sporozoite antigens occur at this site.

The role of CD4+ T cells, and in particular T follicular helper cells in providing help for antibody responses is also a neglected area. Sporozoites can induce CD4+ T cells and numerous CD4+ epitopes have been identified in the CSP proteins of both mice and human malaria strains (Nardin and Nussenzweig, 1993). Moreover, immunization studies with multiple antigen peptides have shown that B cell responses to the NANP repeat are enhanced by the inclusion of T cell epitopes (Tam et al., 1990). CSP-specific CD4+ T cells expressing various effector functions area are also associated with protection by RTS,S (White et al., 2013). Nonetheless the extent to which these cells are acting as direct effectors or through help to antibody responses is unclear.

#### **THE INFLUENCE OF BLOOD STAGE MALARIA ON B CELL RESPONSES TO SPOROZOITES**

One critical factor that may affect the maintenance of sporozoite specific immunity, and immunity to vaccines in general, is malaria infection itself (Urban et al., 1999; Ocana-Morgner et al., 2003). In mouse models, the impact of blood stages on both bystander and malaria-specific immune responses has been examined. *P. yoelii* infection induces apoptosis of memory B cells and plasma cells