Monophosphoryl Lipid-A: A Promising Tool for Alzheimer’s Disease Toll

CHRONIC INFLAMMATION IN ALZHEIMER’S DISEASE: A DOUBLE EDGED SWORD

According to the World Alzheimer Report 2015, the total number of people with dementia worldwide in 2015 was estimated at 46.8 million and is expected to nearly double every 20 years, reaching 74.4 million in 2030 and 131.5 million in 2050. The total number of new cases of dementia each year worldwide is nearly 9.9 million [1]. Alzheimer’s disease (AD), in particular, grows each year at a rate of 4.6 million new cases, with numbers affected doubling every 20 years to reach 81.1 million by 2040 [2]. AD is the major cause of dementia contributing to a huge burden for economics.

As a public health problem, huge efforts are being done in order to develop treatments regarding mostly the prevention, deceleration, and improvement of AD. Drugs that have been tested in AD can be organized into several categories: From natural products to stem cells; however, none has yet proven to change AD natural history [3]. Notably, there is increasing new evidence pointing toward the beneficial pro-inflammatory state in amyloid-β (Aβ) clearance and AD pathological cascade while high inflammatory states facilitate both Aβ production and itsdeposition [4].

One of the most promising research avenues in AD is the cross-talk between inflammation and innate immunity led by Toll-like receptors (TLRs) [5]. These receptors (TLR1 –TLR11) are evolutionary conserved type I transmembrane proteins expressed in both immune and non-immune cells [6]. Relevantly, these are prototypical pattern recognition receptors, which recognize conserved microbial signature molecules known as either pathogen-associated molecular patterns (PAMPs) or microbe-associated molecular patterns. Additionally, TLRs are also endowed with the capacity to recognize an array of endogenous molecules termed danger-associated molecular patterns (DAMPs) [6]. Their ability to initiate and propagate inflammation makes them attractive therapeutic targets in AD [5].

In fact, TLR4 has been implicated in the pathogenesis of AD [7, 8]. Specifically, TLR4 agonist activity not only triggers inflammation pathways in several immune cells including microglia, monocytes, and dendritic cells, thus creating a beneficial pro-inflammatory state, but also is implicated in Aβ clearance, by decreasing the number of Aβ oligomers, altering their constitution to smaller and more soluble molecules [9].

Monophosforyl lipid A (MPL) is a chemically detoxified lipid A moiety derived from Salmonella minnesota R595 lipopolysaccharide (LPS). While LPS is a full TLR4 agonist, MPL is a partial TLR4 agonist [10]. Consistently, this LPS-derived agent exhibits unique immunomodulatory properties at doses that are non-pyrogenic and show low toxicity[9, 11] (please refer to concluding remarks). Therefore, MPL has been studied, both in vitro and in vivo, in AD physiopathology [11]. Additionally, MPL is already being commercialized as a vaccine adjuvant [10]. In particular, it was recently reported that treating transgenic mice overexpressing human amyloid-β protein precursor (AβPP) with a liposomal vaccine containing MPL as an adjuvant (ACI-24; an anti-Aβ vaccine) restored their cognitive impairment [11]. Remarkably, this vaccine entered a combined Phase I/II clinical trial in 2011 which is still ongoing [12].

The role of neuroinflammation and microgliosis in AD is attracting a great deal of attention as recent data suggests that both factors are early events in the pathogenesis of the disease. Particularly, a growing body of evidence has indicated the presence of pro-inflammatory mediators (complement, proteases, cytokines) in the brain and cerebrospinal fluid (CSF) of AD patients [13]. In line with this information, it has been shown that microglial cells, which are the resident macrophages of the central neural system (CNS), play a crucial role in the process of neuroinflammation. In response to cytokines and other signaling molecules from inflammation, microglia transform from a ramified and inactivated state to an activated phagocytic one, releasing pro-inflammatory mediators in the process. In terms of chronic neuroinflammation, these cells can remain activated for extended periods, releasing quantities of cytokines and neurotoxic molecules that contribute to long term neurodegeneration [14].

Astrocytes and microglia express a range of molecules known as “pattern recognition receptors” including TLRs, which are critical for eliciting innate immune responses and to initiate adaptive immunity that activate these cells and initiate a neuroinflammatory reaction [5]. Microglia express the two classes of major histocompatibility complex, MHC class 1 and MHC class 2, and although these antigen presenters are mainly involved in the reaction to infectious disease, they are thought to play a role in the development of neuroinflammation [15].

It has been suggested that pro-inflammatory cytokine levels are related to the magnitude of the plaque burden in the AD brain [4]. Besides, with the role of cyclooxygenase (COX) pathways in neuroinflammation becoming better established, nonsteroidal anti-inflammatory (NSAIDs) drugs have been identified as a class of drug with potential therapeutic effects [16]. Indeed, aspirin (an irreversible COX-1 inhibitor) reduces neuroinflammatory and oxidative insults by reducing prostaglandins and increasing anti-inflammatory lipoxin [16]. However, evidence is lacking for clinical benefit of NSAIDs and selective COX-2 inhibitors in patients with neurodegenerative diseases. The most recent results of a large clinical trial, the Alzheimer disease anti-inflammatory prevention trial (ADAPT), suggest that certain NSAIDs may reduce the chances of an asymptomatic individual developing this disease, but these same drugs exacerbate later stage AD [17].

In fact, during the development of AD pathology, microglia fails to restrict amyloid plaques and may contribute to neurotoxicity and cognitive deficits. Nevertheless, under specific conditions, microglia can participate in cerebral amyloid clearance. This complex relationship between microglia and Aβ pathology highlights both deleterious and beneficial roles of microglial activation in the context of AD [18]. Although a cause and effect relationship between inflammation and AD is yet unveiled, it has been suggested that some components of this complex molecular and cellular machinery are most likely promoting pathological processes leading to AD, whereas other components engage in doing the opposite [19]. Overall, inflammation behaves like a double-edged sword since, on one hand, the inflammatory response facilitates Aβ production and deposition and, on the other hand, it can facilitate Aβ clearance [9].

However, this apparently antagonistic strategy should not be rigidly attributed to specific cytokines under normal and pathological conditions. In fact, the effects of signaling molecules may differ, depending on its location within the CNS and on the context of disease [4].

THE IMPORTANCE OF INNATE IMMUNE SYSTEM IN AD-RELATED PATHOLOGY

The innate immune system that relies strongly on the microglia (resident macrophages in the brain) has a major role in AD [20]. While neglected for decades, neuroinflammatory processes coordinated by microglia are now accepted as etiologic events in AD evolution [18]. Innate immune system implications are best described with the attraction process of microglia into diseased brain regions being often mediated by the interaction of chemokines and cytokines with their receptors, which are both expressed by microglial cells. In the context of AD, several lines of evidence support the notion that amyloid-β (Aβ) plaque associated factors, including misfolded Aβ peptides themselves, act as microglial attractants [21]. Cytokines have also been suggested to be involved in chemoattraction of microglia to amyloid lesions [4]. Studies with Cx3cr1 knockout transgenic mice [22] suggested that increased pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), IL-1α, and granulocyte macrophage-colony stimulating factor are related to microglia attraction [23]. Once microglia moves toward Aβ deposits, these cells express various cell surface receptors allowing them to recognize and interact with misfolded Aβ peptides. In this context, the scavenger receptors have garnered attention, as they can bind diverse ligands and affect the activation level, inflammatory status, and phagocytic function of microglia [24]. Bamberger and collaborators (2003) described a receptor complex including CD36, the integrin-associated protein CD37 and the α6β1 integrin, which interacts with Aβ fibrils and activates microglial secretion of reactive oxygen species [25]. Another key scavenger receptor, the receptor for advanced glycation end products, has also been identified on CD68+ microglial cells close to senile plaques in AD patient brains and in cultured microglia from rat and mouse models of the disease [18]. Remarkably, TLRs and associated receptors (e.g., CD14) are highly expressed by microglia in close proximity to plaques in AD patient brains and in mouse models of the disease [18].

TOLL-LIKE RECEPTORS: MOLECULAR STRUCTURE, CELL DISTRIBUTION, AND FUNCTIONS

Toll-like receptors are evolutionarily well conserved type I transmembrane proteins on the surface of both immune and non-immune eukaryotic cells [5]. These receptors comprise a N-terminal leucine-rich repeats which is the extracellular binding domain and a highly conserved C-terminal domain termed the TIR - Toll/interleukin (IL)-1 receptor domain [26]. Microglia express all TLRs identified to date, whereas astrocytes, oligodendrocytes, and neurons express a more limited TLR repertoire [6].

The ligands for these pattern recognition receptors (TLRs 1 to 11) are components of pathogenic microbes and are often called PAMPs, including bacterial cell wall components; bacterial genome DNA and viral, fungal, and parasitic products [21]. TLR immune system is also concerned with damage signals from injured tissue. In fact, it is suggested that TLRs recognize not only PAMPs, but also stress/damage or DAMPs [7]. In general, most DAMPs are the consequence of cell death, necrosis, or tissue remodeling and include mammalian genomic DNA, high-mobility group box 1 protein (HMGB1), heat shock proteins (HSPs: HSP22, HSP60, HSP70, HSP96), extracellular matrix products (e.g., hyaluronan, type III repeat extra domain of fibronectin), uric acid crystals (urates), β-defensin and, finally, plant ligands (paclitaxel) are also known TLR ligands (Table 1) [27].

TLR signaling triggers the transcription of nuclear factor for the kappa light chain enhancer in B cells (NF-κB) and activating protein-1 (AP-1), which plays a crucial role in inflammation through encoding important pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6, IL-8, and IL-12 [13]. TLR 3 and TLR 4 also activate the production of type 1 (α and β) interferons by inducing the transcription factors interferon regulatory factors 3 and 7 (IRF3 and IRF7) [5]. Notably, it was demonstrated that fibrillar Aβ_1 - 42 may act as a PAMP-like infectious agent through TLR activation [26]. This is highly suggestive that TLRs may be a potential therapeutic target in AD.

Besides inflammation, TLRs also exert determinant functions related to the processes of neurogenesis, learning and memory in the absence of any underlying inflammation/infectious etiology [27].

TLR4 MODULATION IN AD

Activation of TLRs on microglia may modulate AD progression [13]. On one hand, investigators observed that a deficiency of TLR2 and TLR4 in cultured microglia significantly reduced Aβ-triggered inflammation [7]. Moreover, it was alreadydemonstrated that Aβ triggers microglial inflammatory mediator production, mainly via TLR4, but also via TLR5 and TLR6 [6]. On the other hand, stimulation of microglial cells with TLR2, TLR4, or TLR9 specific agonists boosts Aβ clearance both in vitro and in vivo. Additionally, microglia deficient in TLR2, TLR4, or the co-receptor CD14 are not activated by Aβ and do not exhibit a phagocytic response [5]. Moreover, transgenic AD mice lacking TLR4 have markedly elevated levels of diffuse and fibrillar Aβ [3]. Additionally, the TLR4-deficient mice also showed to have elevated TNF-α, IL-1β, IL-10, IL-17, CD11b, and GFAP (glial fibrillary acidic protein) levels, further suggesting that TLR4 signaling is involved in AD natural history [28]. Altogether, TLR4 signaling shows a functional dichotomy: Having a beneficial role in clearing the Aβ deposits or being prejudicial by triggering a high inflammatory cascade in the AD diseased brain as elicited before [7]. The balance is tipped not only by the AD status but also by the TLR4 genetic background and by the level of inflammation produced by TLR4activation [29].

Two different pathways are activated via TLR4 signaling, leading to the induction of inflammation: One involves the recruiting of the myeloid differentiation primary-response gene 88 (MyD88) to activate the NF-κB, p38, and JNK/MAPK pathways via TRAF6 [30]; the other occurs in the presence of CD14, independently of MyD88 [26]. Recent studies highlighted that these MyD88-dependent and MyD88-independent pathways could be also engaged in upregulation of phagocytic gene expression programs in AD [31]. However, relatively less attention has been devoted to microglial phagocytosis.

3-O-DESACYL-4’-MONOPHOSPHORYL LIPID A

3-O-desacyl-4’-monophosphoryl lipid A (MPL) is obtained from Salmonella Minnesota R595 LPS and is a LPS-derived TLR4 agonist that exhibits unique immunomodulatory properties at doses that are nonpyrogenic [5, 32].

Thus, MPL acts as an immunostimulant and is part of already licensed or candidate vaccines [33]. In particular, MPL directly affects the innate immune response, orchestrating the quality and intensity of the adaptive immune response to the vaccine antigen [34]. The incorporation of MPL in different adjuvant formulations forms the basis of Adjuvant Systems (AS) that have been put in place since 1990 [10]. For example, MPL forms the basis for Adjuvant System 04 (AS04), when combined with aluminum salts [35]. This aluminum salt, and TLR4 agonist, based adjuvant system retains its ability to activate TLR4 and stimulates most of the innate immune response [35]. Furthermore, the AS04-induced innate responses were primarily due to MPL [35].

By now, ACI-24 is the only vaccine being studied in the context of AD that utilizes MPL as adjuvant [19].

RECENT EXPERIMENTAL EVIDENCE LINKING MPL AS A DISEASE MODIFYING AGENT IN AD NATURAL HISTORY

Pre-clinical and clinical studies have been carried out to test the TLR4 stimulation with the detoxified ligand MPL in the improvement of AD-related pathology. This molecule was used either as a vaccine adjuvant to AD immunotherapy with active Aβ vaccination or by itself as a single compound.

Aβ vaccination is an approach under investigation to prevent and/or treat AD.

Maier and colleagues affirmed that successful active immunization with Aβ_40/42 vaccination requires a strong and safe adjuvant to induce anti-Aβ antibody formation [36]. In particular, MPL is a potent adjuvant, being useful in breaking tolerance to self-antigens such as Aβ. Herein, the authors compared the adjuvants MPL/trehalose dicorynomycolate (TDM), cholera toxin B subunit (CTB), and Escherichia coli heat-labile enterotoxin LT(R192G) for their ability to induce a humoral and cellular immune reaction, using fibrillar Aβ_40/42 as a common immunogen in wildtype B6D2F1 mice. On one hand, MPL/TDM [subcutaneous administration (s.c.)] triggered anti-Aβ antibodies levels that were up to four times higher compared to s.c. LT(R192G) (Fig. 1). On the other hand, weekly intranasal (i.n.) administration over 11 weeks of Aβ_40/42 with CTB induced only moderate levels of antibodies. This is in contrast with i.n. immunization with Aβ_40/42 + CBT that generated high levels of anti-Aβ antibodies. All immunogens generated antibodies that recognized mainly the Aβ_1–7 epitope and specifically detected amyloid plaques on AD brain sections. The subclass of immunoglobulin that is induced after immunization is an indirect measure of the relative contribution of Th2-type cytokines versus Th1-type cytokines. For example, the production of IgG1-type antibodies is primarily induced by Th2 cytokines, whereas production of IgG2a-type antibodies reflects the involvement of Th1-type cytokines. Importantly, anti-Aβ antibodies induced by MPL/TDM were mainly IgG2b, IgG1, and lower levels of IgG2a and IgM. Additionally, splenocyte isolated from MPL/TDM animals showed a moderate proliferation and IFN-γ production in vitro following Aβ_40/42 stimulation. In contrast, LT(R192G), generated predominantly IgG2b and IgG1 anti-Aβ antibodies with very low splenocyte proliferation and IFN-γ production. Antibodies produced by Aβ_40/42 with CTB were not further characterized. Table 2 details the humoral and cellular profile of the elicited response to the immunization protocol, combining in vivo (Ig subclass and IgG1/IgG2 ratio; plasma samples) and in vitro (stimulation index and Th1-type cytokines versus Th2-type cytokines; splenocytes) data presented in Maier et al. [36]. Importantly, the Maier et al. [36] study showed no signs of inflammation or toxicity of immunized mice.

Along the premises already held by Maier et al. [36] that immunization against Aβ is a promising approach for the treatment of AD before its clinical onset, Kofler and colleagues immunized non-human primates with the combination of Aβ₄₂ admixed with MPL [37].

In detail, ten non-human primates (NHP) of advanced age (18–26 years) and eight 2-year-old juvenile NHPs were immunized at 0, 2, 6, 10, and 14 weeks with aggregated Aβ₄₂ admixed with MPL as adjuvant, and monitored for up to 6 months. Aβ oligomer composition, anti-Aβ antibody levels, and immune activation markers, including microglial activity, were assessed in plasma, CSF samples, and brain regions, before and at several time-points after immunization. Microglial activity was determined by (¹¹C) PK11195 (a selective radioligand of the peripheral benzodiazepine receptor) PET scans acquired before and after immunization, and by postmortem immunohistochemical and real-time PCRevaluation.

Kofler et al. [37] concluded that all juvenile animals showed a strong and sustained serum anti-Aβ IgG antibody response, while only 60–80% of aged animals developed detectable antibodies as detailed in Table 3. Moreover, the immune response in aged monkeys was more variable between animals, delayed and significantly weaker when compared with the juvenile group. On the other hand, all juvenile animals contained anti-Aβ antibodies in the CSF at week 16, when serum antibody concentrations were at peak levels. However, CSF levels were much lower than serum levels. By contrast, anti-Aβ antibodies were only seen in 22–44% of the aged animals (Table 3). Taken together, the PET, immunohistochemistry, and PCR data indicate that vaccination against Aβ was not associated with significant microglial activation of aged animals. The juvenile immunized animals were not euthanized. Additionally, the immunization protocol did not induce increased inflammation, neither of the autoimmune/encephalitic type nor of the innate arm of the immune system. Moreover, average Aβ₄₀, Aβ₄₂, and tau plasma and CSF levels were not significantly different between pre- and post-immunization time points for either aged or juveniles (Table 4). Furthermore, pre-immunization baseline for plasma and CSF levels were not significantly different between juvenile and aged animals (Table 4). Notably, a shift in the composition of soluble oligomers toward smaller species was observed in the aged animals, although no effect on overall brain Aβ levels was seen. Finally, Kofler et al. [37] concluded that preventive Aβ immunization is a safe therapeutic approach without serious side effects in both aged and juvenile NHPs. Recently, Michaud et al. tested MPL, not as a vaccine adjuvant (as in the previous studies), but as a therapeutic molecule by itself in AD-related pathology, producing an exciting and promising study [32].

These authors conducted both in vitro and in vivo studies. Microglia cells showed cytoskeletal remodeling and a significant increase in the internalization of Aβ₄₂ oligomers upon LPS or MPL stimulation, although at very different levels: While MPL only induced a moderate pro-inflammatory response in microglia but a strong microglia phagocytic response, LPS triggered both phagocytic and strong pro-inflammatory status. In fact, MPL did not increase COX-2 and nitrite levels, nor triggered the expression of cytokine-inducible nitric oxide synthase. However, it induced both TNF-α and CCL2 mRNA expression, although at lower levels when compared to LPS. No differences between both drugs were noticed in cell migration.

Michaud and colleagues [32] also found that there was a smaller increase in the levels of cytokines (TNF-α and IL-6) and chemokines (CCL3, CXCL-1,and IP-10) measured in the sera of wild-type C57BL/6 mice following a single intra-peritoneal (i.p) injection of MPL when compared to LPS-injected mice. This moderate inflammatory response is consistent with the in vitro data. Moreover, these authors showed that both MPL and LPS triggered a strong and similar monocytopoiesis following i.p. administration when compared with controls as measured by flow cytometry.

To investigate whether treatment with MPL might affect AD-related pathology in vivo, Michaud et al. [32] administered MPL, LPS, or PBS (controls) weekly by i.p. injection to APPswe/PS1 mice for 12 consecutive weeks beginning when the mice were3 months old. These double transgenic mice develop age-associated amyloid pathology and deficits in hippocampal-dependent memory, being a very sought-after AD animal model [38]. Cognitive function and Aβ deposition were assessed for each mouse 2 and 3 week after the final injection, respectively. MPL-treated APP_swe/PS1 mice showed significant improvement in cognitive function in the T water maze behavioral test, compared with the PBS-treated control group. LPS treatment, however, did not lead to significant improvement in the cognitive performances of these mice. Additionally, MPL caused a significant reduction in the number and size of Aβ deposits, as well as the quantity of soluble Aβ in the brain. These authors further demonstrated that phagocytosis of Aβ was responsible for its clearance as levered by higher percentages of Aβ-positive CD45+ brain cells in the MPL-group compared to the control group. On the contrary, the size and number of Aβ plaques were considerably greater in LPS treated animals, whereas the level of soluble Aβ monomers was equivalent compared with controls. Overall, Michaud et al. [32] demonstrated that MPL induced a potent phagocytic response by microglia while triggering a moderate inflammatory reaction. Importantly, these authors showed that clearance of Aβ correlated with improved cognitive functions in MPL-treated APP_swe/PS1 mice as summarized in Table 5.

ACI-24 (AC Immune^®, Lausanne, Switzerland) is an Aβ_1 - 15 peptide to which two lysines that are tetrapalmitoylated on the ɛ-nitrogens were attached to both ends. The antigen is embedded in a liposome membrane. A mixture of the lipids DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DMPG (1,2-Dimyristoyl-sn-Glycero-3-PhosphoGlycerol), cholesterol, and MPL in a ratio of 9:1:7:0.06, respectively was used as adjuvant [19]. Pihlgren et al. showed that ACI-24 vaccine induced anti-Aβ antibody titers in the APPxPS1 transgenic mice with high specificity for oligomeric Aβ species [39]. These antibodies were of IgG2b and IgG3 isotypes, indicating a preferential Th2 vaccine response. The brain tissues showed no evidence of microglia activation nor astrogliosis and no increase in the levels of the pro-inflammatory cytokines IL-1β, IL-6, TNF-α, or IFN-γ [36]. The histological staining with Perl’s iron revealed that the number of sections per mouse containing large hemorrhages was significantly lower in mice treated with ACI-24 compared to the vehicle-treated controls [39]. Additionally, ACI-24 immunization did not induce infiltration of peripheral monocytes (MHC-II), or of peripheral T- and B-cells (CD4 or CD45) into the brains of the treated transgenic mice [39]. The results show that immunization of old transgenic mice withACI-24 induces oligo-specific anti-Aβ antibodies and decreases the number of large micro-hemorrhages without inducing micro-hemorrhages [40]. Furthermore, ACI-24 immunization does neither cause cellular brain inflammation nor enhance the release of pro-inflammatory cytokines, nor does it result in the penetration of peripheral monocytes into the brain. These results, together with the preferentially Th2 associated antibody isotype profile, indicate a low risk of encephalitis and thus demonstrate a positive safety profile for ACI-24 in a relevant AD animal model [39].

ACI-24 was evaluated in a Phase I/II clinical trial in Denmark, Finland, and Sweden. The phase I/II double-blind, randomized, placebo-controlled, adaptive design study of the safety, tolerability, immunogenicity and efficacy of ACI-24 in patients with mild to moderate AD started on 2011 and is still ongoing [12].

The overall study objective is to assess the safety, immunogenicity, and efficacy of repeated doses of ACI-24 at 3 different dose levels administered to patients with mild to moderate AD patients [12].

The secondary objectives of this trial are to explore the following parameters: The efficacy of ACI-24 in reducing Aβ level in the brain of patients with mild to moderate AD; the effect of ACI-24 on whole brain and hippocampal volume; the effect of ACI-24 on T cell activation; the effects of ACI-24 on putative biomarkers of the progression of AD including total tau and phosphorylated tau protein (phosphotau) and Aβ levels (Aβ₄₂ and Aβ₄₀) in blood and CSF; the efficacy of ACI-24 on clinical end points in patients with mild to moderate AD [12].

Primary end points refer to (1) safety and tolerability assessments: Adverse events; global assessment of tolerability; physical, neurological and psychiatric examination; vital signs; MRI imaging; electrocardiogram; routine hematology and biochemistry evaluation in blood and urine; inflammatory markers in blood and CSF; (2) biological assessments: Immune response titer (serum anti-Aβ₄₂ IgG titer); and finally, (3) efficacy assessments: Change from baseline over 1 year of total cognitive score (Neuropsychological Test Battery) [12].

The subjects will be treated for approximately 12 months (treatment period) and will enter a follow-up of 24 months (follow-up period). There will be 198 individuals studied, 58 from ages 18 to 65, and 140 above 65 years old, both male and female [12]. ACI-24 is still under evaluation. Table 6 summarizes main conclusions from the pre-clinical and clinical studies presented herein.

OTHER ADJUVANTS IN AD

Other adjuvants that reached clinical development in AD are presented and put in perspective regarding MPL.

The first anti-Aβ vaccine (AN1792) tested in AD patients included full length Aβ_1 - 42 peptide with an adjuvant (QS21) that preferentially promoted T-cell-mediated immune responses [41]. This is a saponin derivative of MPL [42]. However, based on the clinically significant adverse events and questionable clinical efficacy, the vaccine was abandoned [41].

Another two adjuvants are being tested in Aβ immunotherapy and reached clinical development. CAD106, which is undergoing Phase II/III clinical trials [41], combines multiple copies of Aβ_1 - 6 peptide derived from the N-terminal B cell epitope of Aβ, coupled to a Qβ virus-like particle (VLP). Qβ-VLPs are composed of 180 monomeric capsid protein subunits of 14 kDa each [43] that can readily be produced in E. coli, where the monomers of Qβ self-assemble into a 25 nm icosahedral capsid in the citosol. During expression, E. coli RNA gets packaged inside the Qβ particles and it serves as a potent agonist for TLR7/8 [44, 45]. This is an active vaccination strategy that aims to elicit a strong antibody response while avoiding inflammatory T cell activation [46]. In fact, CAD106 induced Aβ-antibody titers in APP-transgenic mice and monkeys without activating Aβ-reactive T cells in mice. The antibodies effectively interfered with amyloid accumulation in APP transgenic mice brain and protected from Aβ toxicity [47]. This vaccine shares with MPL the ability to reduce Aβ accumulation without eliciting a strong adverse inflammatory response. However, whether Qβ virus-like particle targets TLR4 is virtually unknown. On the other hand, the E. coli RNA that is packaged inside the Qβ particles serves as a potent agonist for TLR7/8 [44, 45]. Moreover, the VLP carrier provides both a scaffold for ordered presentation of the antigen and T-helper cell epitopes [48].

Aluminum hydroxide is another adjuvant, which is coupled to the smaller Aβ immunogens (Aβ_1 - 6) in the Affitope AD02 [41]. This immune therapy may allow for the production of anti-Aβ antibodies while minimizing a pro-inflammatory TH1 response. Clinical data from Phase I trials confirmed for this vaccine positive antibody responses with no signs of adverse autoimmune inflammation [49]. This vaccine is under phase II trial. It is accepted that aluminum salts (aluminum hydroxide, aluminum phosphate) adjuvant activity is twofold: Promoting antigen uptake and stimulation of innate immunity at the injection site. However, it remains unclear how aluminum activates innate immunity and which innate immune receptors are responsible for its adjuvanticity [50].

CONCLUSION

Herein we review evidence suggesting that finely tuning the innate immune system may be instrumental in AD therapeutics [51]. In fact, Michaud et al. [32] showed that whereas chronic systemic LPS administration in APP_swe/PS1 mice exacerbated the Aβ plaque load due to its strong TLR4 agonist activity and pro-inflammatory profile, MPL— a derivative of the lipid moiety of lipopolysaccharide, which also targets TLR4— decreased Aβ load including decreasing the quantity of soluble Aβ in the brain and improved cognition in this AD mouse model while triggering a modest inflammatory response [32]. These authors suggested that the underlying mechanism could be microglial activation through TLR4 activation. However, whether MPL crosses blood-brain barrier (BBB) does not clearly stem from Michaud et al. [32]. Alternatively, these authors offered the following two possibilities to explain how peripheral administration of MPL has impact to brain: 1) MPL activates peripheral Aβ phagocytosis by blood monocytes. This could contribute to brain Aβ load reduction through equilibrium-driven redistribution of Aβ to periphery [52]; 2) MPL may promote mobilization of microglial precursors from bone marrow into the brain perivascular spaces thus directly phagocyting Aβ from brain [53]. Although these mechanisms could be partially responsible for Aβ brain clearance, they certainly cannot explain how MPL could induce a potent phagocytic response by brain resident microglia as suggested by Michaud et al. [32]. One should bring to discussion that there is novel evidence showing that the AD mouse model APP/PS1 has increased BBB permeability [54]. Therefore, MPL likely crosses a perturbed BBB, thus having a direct impact in brain resident microglial cells. Moreover, increased proportion of CD45 +cells containing Aβ found in the brain could also be infiltrating monocytes/macrophages that were already reported by Minogue et al. [54]. Finally, a perturbed BBB might facilitate a crosstalk between peripheral and central innate immune system [55]. Overall, the authors further concluded that compounds that sparingly stimulate innate immune system may have a great AD therapeutic potential. Specifically Aβ modifications are clearly related to AD pathology since soluble oligomers are increased in human AD brain tissue. Moreover Aβ soluble oligomers are more strongly associated with synaptotoxicity and cognitive impairment than fibrillar Aβ [51]. Notably, toxicity has been attributed to dimers, but also to trimers, pentamers, and dodecamers [56].

Aging is concurrent with diminished function of both the adaptive and innate arms of the immune system [57]. This includes an increased threshold for immune response induction, which may be overcome by supplementing a vaccine with an appropriate immunostimulatory adjuvant [58]. Interestingly, MPL is being used in humans safely as a vaccine adjuvant in approved vaccines against human papillomavirus (HPV) and hepatitis B (HBV); and is being evaluated in candidate vaccines against malaria, HIV (human immunodeficiency virus), tuberculosis. HPV and HBV vaccines are licensed worldwide and in Europe, respectively [34].

Notably, there is a growing amount of studies confirming the immunostimulatory vaccine adjuvant characteristics of MPL when using active anti-Aβ immunization strategies, in AD related pathology [37].

In particular, the vaccination route (subcutaneous or intra-nasal) had impact on anti-Aβ antibodies production in immunized mice with Aβ peptides+MPL [36]. In fact, the MPL oil-in-water emulsion used for subcutaneous immunizations did not generate a significant amount of antibody titer when used intranasally [36]. This might be due to less absorption of this emulsion by the mucosal epithelia and/or to differences in the antigen processing immune cells at the two locations (e.g., dendritic cells for subcutaneous immunization versus M cells in the nasal epithelium and lymphocytes and dendritic cells in the nasopharyngeal-associated lymphoreticular tissue for intranasal immunization) [36]. In this review, it is also highlighted that the number of immunizations may also influence the immune response triggered by Aβ+MPL strategy, besides route of administration. Therefore all these parameters must be taken in consideration when designing clinical trials using MPL as an adjuvant when combined with Aβ.

MPL was also tested as adjuvant in Aβ preventive immunotherapy in NHP [37]. This model has close genetic relationship to humans, identical APP amino acid sequence, and natural development of neuritic amyloid plaques and amyloid angiopathy with age. However, there are also differences between AD patients and NHP models, such as less severe tau pathology and significant differences in CSF biomarkers. Nonetheless, it was demonstrated that this preventive Aβ immunization triggered a significant shift in oligomer size with an increase in the dimer/pentamer ratio in aged animals compared with non-immunized controls. This might facilitate removal of toxic Aβ species from the brain, thus tipping the balance between Aβ accumulation and clearance toward the latter.

Altogether, this study strongly suggested that preventive Aβ immunization using MPL as an adjuvant is a safe therapeutic approach lacking adverse CNS immune system activation. Along these lines, a vaccine using MPL as an adjuvant (ACI-24) is currently undergoing a phase I/II clinical study in patients with mild to moderate AD.

Overall, this review stresses that MPL holds a great potential as safe and effective treatment for the most common neurodegenerative disease worldwide— AD. On the other hand, MPL, besides adjuvant, can be formulated to act as new therapeutic in AD by itself.

CONCLUDING REMARKS

Schwartz et al. suggested that a tightly regulated stimulation of innate immune instead of its complete inhibition is a promising way of designing new treatment options for AD [59]. On the other hand, the recognition of the involvement of microglial TLR4 in AD pathogenesis strongly suggests that it may be an appropriate therapeutic target for AD. Along these lines, MPL, which is a TLR4 agonist, is a promising therapeutic option for AD treatment. In fact, this drug is endowed with the ability to upregulate phagocytic microglia phenotype, thus promoting Aβ clearance without eliciting a strong adverse inflammatory response. These translated into an improvement of AD related pathology in an AD mouse model. There is a striking feature regarding MPL that was highlighted by Bryant et al.: MPL is a partial agonist [10]. This means that MPL binds to TLR4 and induces some conformational change without leading to full activation of this receptor, thus resulting in incomplete signaling. Specifically, in opposition to the lipid A moiety of LPS, MPL lacks the phosphate group at position 1 and is unable to contact positively charged residues on the surface of both MD2 and TLR4 [60]. This is consistent with MPL stimulating microglial phagocytic signaling pathways thus promoting Aβ uptake without extensive stimulation of the proinflammatory pathways (Fig. 2).

Safety issues have also to be tackled prior to clinical use of a novel therapeutic strategy. Importantly, MPL safety was not confirmed in humans. Nonetheless, MPL is already FDA-approved in humans as an adjuvant in different vaccines and ACI-24 study is still ongoing. Therefore, MPL safety is likely and clinical application can be accelerated. There are still open questions that need to be answered to further facilitate the understanding of the putative therapeutic benefits of MPL as a stand alone therapy: How does MPL affect other hallmarks of AD pathology such as dystrophic neurites and hyperphosphorylated tau aggregates? Does MPL cross BBB? What is MPL mechanism of action including its impact in neuronal and astrocytic TLR4?

Overall, we argue that the TLR4 partial agonist MPL is a potentially safe and effective new single pharmacological tool in AD.