domain: chim sdv

text:
Jordi Molgó
email: jordi.molgo@inaf.cnrs-gif.fr

Evelyne Benoit

Rómulo Aráoz

Armen Zakarian

Bogdan I Iorga
email: bogdan.iorga@cnrs.fr

SPIROLIDES AND OTHER CYCLIC IMINE TOXINS: TOXICOLOGICAL PROFILE

In this chapter, we review available evidence on the toxicological profile of spirolides and other lipophilic cyclic imine toxins, highlighting their chemical structure, the phytoplankton species involved in their production, their pharmacokinetics/toxicokinetics and experimental toxicity, and their molecular targets and mechanisms of action. These phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Their chemical structure is represented by a macrocycle, with the ring size between 14 and 27, and two conserved features that include the cyclic imine group and spiroketal ring system. The producers of spirolides, gymnodimines and pinnatoxins have been identified as being the dinoflagellates Alexandrium ostenfeldii/peruvianum, Karenia selliformis and Vulcanodinium rugosum. Their acute toxicity, appraised by the mouse bioassays, classifies them as "fast-acting" toxins because they induced rapid onset of neurological symptoms followed by death within a few minutes. The spirolide congeners are the most toxic after intraperitoneal injection, while there are indications that pinnatoxins are the most toxic group after oral administration. The neurotoxic effects reported for these phycotoxins are mostly due to their specific interaction with the muscle-and neuronal-types of nicotinic acetylcholine receptors which are the principal molecular targets of spirolides, gymnodimines and pinnatoxins, so far studied. Hence, these phycotoxins exhibit both high affinity and broad specificity on nicotinic receptors, indicating that their sites of interaction in the receptors include amino acid residues highly conserved among animal species.

INTRODUCTION

Spirolides were first discovered in the 1990s from the Atlantic coast of Nova Scotia in Canada when unusual toxicities were detected by mouse bioassay in mussel and scallop extracts. Since then, the spirolides have been found to have a worldwide distribution, with a remarkable diversity in their chemical profiles. Spirolides belong to the so-called cyclic imine group of neurotoxins that also include the gymnodimines, pinnatoxins, pteriatoxins, portimine, spiro-prorocentrimine and prorocentrolides (for a recent review, see [START_REF] Stivala | Synthesis and biology of cyclic imine toxins, an emerging class of potent, globally distributed marine toxins[END_REF]. The generic chemical structure of the cyclic imine toxins is represented by a macrocycle, with the ring size between 14 and 27, and two conserved features that include two subunits: the cyclic imine group (which in most cases is found as a spiroimine) and the spiroketal ring system. The cyclic imine, which has been shown to be a key pharmacophoric feature, can be a 5-membered (portimine), 6-membered (gymnodimines, spiroprorocentrimine, prorocentrolides) or 7-membered (spirolides, pinnatoxins, pteriatoxins) ring, that in many cases is substituted with one or two methyl groups. The spiroketal ring system can be a simple tetrahydrofuran (i.e. portimine, gymnodimine) or tetrahydropyran group (i.e. spiroprorocentrimine, prorocentrolides), but also a more complex 6,5-(spirolides H and I), 6,6,5-(spirolide G), 6,5,5-(spirolides A-F) or 6,5,6-spiroketal (pinnatoxins, pteriatoxins). For each sub-family, the first member is shown and the positions for which variations in the substitution pattern were reported are numbered and highlighted in orange. The characteristic spiroimine and cyclic imine features are colored in blue and magenta, respectively, and the size of the macrocyclic ring system in each sub-family is shown in magenta.

Spirolides and the cyclic imine toxins studied so far are "fast-acting" phycotoxins, because of the rapid beginning of neurological symptoms and the short time to death. The basis for these neurotoxic effects is in direct relation to their molecular antagonist action on nicotinic acetylcholine receptors. In this chapter, we summarize our current understanding of the toxicological profile of spirolides and other cyclic imine toxins, with emphasis on their chemical structure, producing organisms, pharmacokinetics/toxicokinetics, experimental toxicity, molecular targets and mechanism of action.

SPIROLIDES -Chemical characteristics of spirolide toxins

Spirolides constitute the largest family among cyclic imine toxins. As shown in Figure 2, to date sixteen spirolide analogues have been reported, which can be classified into four distinct groups according to the structure of the spiroimine and spiroketal ring systems. The first group of spirolides has 10 members with a characteristic 6,5,5-spiroketal ring system. As compared to spirolides A and B, the spirolides C and D and their analogues have an extra methyl group at the position 31 on the imine ring, which is believed to render them more resistant to enzymatic and acidic hydrolysis [START_REF] Christian | Are spirolides converted in biological systems?-A study[END_REF]. Spirolides A and C and their analogs have a double bond between the positions 2 and 3 on the lactone ring which is not present in the structure of spirolides B and D and their derivatives. Other positions where structural variability was observed for spirolides C and D are 13 and 19 on the spiroketal ring system, with the presence or absence of methyl substituents, and 27, where hydroxy and keto substituents were evidenced in some cases. The second spirolide group contains the biologically inactive spirolides E and F, which present the same chemical features as spirolides A and B, respectively, except for the cyclic imine that becomes an open ring amino ketone (reviewed by [START_REF] Guéret | Spiroimine shellfish poisoning (SSP) and the spirolide family of shellfish toxins: Isolation, structure, biological activity and synthesis[END_REF]. These structural changes suggest that they are probably shellfish metabolites of spirolides A and B. The third spirolide group, represented by spirolide-G and 20-methyl spirolide-G, has a 6,6,5-spiroketal ring system with a hydroxyl group at C17 for which the stereochemistry could not be determined [START_REF] Mackinnon | Spirolides isolated from Danish strains of the toxigenic dinoflagellate Alexandrium ostenfeldii[END_REF]) and a methyl substituent at C20 that is present only in the second member of this group. The rest of the structure is identical with spirolide-C, with the dimethyl substituted 7-membered cyclic imine and the unsaturated lactone ring. The fourth spirolide group includes spirolides H and I whose structures are identical to spirolides C and D, respectively, apart from the presence of a 6,5-instead of the 6,5,5spiroketal ring system [START_REF] Roach | Characterization of a dispiroketal spirolide subclass from Alexandrium ostenfeldii[END_REF]. Spirolide-H is the 2,3-unsaturated counterpart of spirolide I.

-Spirolide producing organisms

The producers of spirolides and other cyclic imine toxins were identified as dinoflagellates, a large and diverse group of marine eukaryotic microalgae known to be the major causative agents of harmful algal blooms. Although many dinoflagellate species can produce various natural toxins that impact humans, those involved in the production of Cyclic Imine toxins are limited to mainly Alexandrium, Karenia (formely Gymnodinium), Vulcanodinium and Prorocentrum species (for reviews, see Molgó et al., 2014a;[START_REF] Stivala | Synthesis and biology of cyclic imine toxins, an emerging class of potent, globally distributed marine toxins[END_REF].

All known spirolides have been so far linked to Alexandrium species, and differences in spirolides content and profiles have been reported for dinoflagellate strains from different coastal regions.

Hence, the globally distributed species Alexandrium ostenfeldii [START_REF] Cembella | Spirolide composition of micro-extracted pooled cells isolated from natural plankton assemblages and from cultures of the dinoflagellate Alexandrium ostenfeldii[END_REF] was formerly identified as the biological source of all known spirolides (Table 1). However, during the past six years, another dinoflagellate species, Alexandrium peruvianum, was also recognized as the producing organism for some spirolide congeners in Northwestern Mediterranean Sea waters, Irish coastal waters [START_REF] Touzet | Morphogenetic diversity and biotoxin composition of Alexandrium (dinophyceae) in Irish coastal waters[END_REF] and in the United States coastal waters (see Table 1). It is worth noting that spirolides E and F were not detected in dinoflagellates, but were attributed to be metabolically modified products present in shellfish [START_REF] Hu | Characterization of biologically inactive spirolides E and F: Identification of the spirolide pharmacophore[END_REF].

Table 1 near here -Pharmacokinetics/toxicokinetics of spirolide toxins

Among the spirolides, both 13-desmethyl spirolide-C and 13,19-didesmethyl spirolide-C have been shown to cross a Caco-2 cell monolayer [START_REF] Espiña | 13-desmethyl spirolide-c and 13,19didesmethyl spirolide-c trans-epithelial permeabilities: human intestinal permeability modelling[END_REF]. Caco-2 trans-epithelial permeability assays represent a good model to predict the intestinal absorption of drugs and toxins. When administered orally with a single non-lethal dose to mice deprived of food (24 hours before, but given a nutritive solution with salts and glucose) both spirolides were detected in blood and urine samples after 15 minutes and 1 hour of administration, respectively as revealed by liquid chromatography-tandem mass spectrometry [START_REF] Otero | Pharmacokinetic and toxicological data of spirolides after oral and intraperitoneal administration[END_REF]. The 13-desmethyl spirolide-C and 13,19-didesmethyl spirolide-C were also detected 1 hour after intraperitoneal (i.p.) administration in urine, and trace amount of both toxins in blood; after 24 hours important amounts were detected in feces. These results indicate that spirolides are absorbed when administered orally, and are excreted through the urine and feces. The histological evaluation by light microscopy of the heart, kidneys, liver, spleen, stomach, and intestines, performed 24 hours and 7 days after oral administration, did not reveal any observable alteration [START_REF] Otero | Pharmacokinetic and toxicological data of spirolides after oral and intraperitoneal administration[END_REF]. Further studies, using ultra-performance liquid chromatography-mass spectrometry and proton magnetic resonance spectroscopy, have shown that 13-desmethyl spirolide-C is found in the brain a few minutes after its intraperitoneal injection to 3xTg mouse (a well-known model of Alzheimer's disease), and remains measurable even 24 hours post-administration, indicating that the spirolide traverses the blood brain barrier.

Furthermore, a decrease in the intracellular amyloid beta levels in the hippocampus of treated 3xTgmice was observed, emphasizing the positive effects of the molecule in this mouse model [START_REF] Alonso | Benefit of 13-desmethyl spirolide C treatment in triple transgenic mouse model of Alzheimer disease: beta-amyloid and neuronal markers improvement[END_REF].

-Toxicity of spirolide toxins

Although in vitro cytotoxicity tests have been used, the most appropriate assays for appraising the toxicity of Cyclic Imine toxins and estimating their risk to human health are in vivo methods.

Hence, to date, most if not all studies regarding the acute toxicity of cyclic imines have been performed using mainly the mouse bioassays -with synthetic toxins, or toxins extracted from either cultured dinoflagellates or contaminated shellfish, administrated to animals by i.p. injection, gavage administration, or voluntary consumption (feeding) -and determining their 50% lethal dose (LD 50 ) values. It is worth noting that cyclic imine oral administration, with feeding taking priority over gavage, is a more relevant method since, first, it closely reproduces the situation in humans, and second, it avoids the use of high concentrations of solvent (i.e. ethanol) to dissolve the toxins (for reviews, see Molgó et al., 2014a;[START_REF] Stivala | Synthesis and biology of cyclic imine toxins, an emerging class of potent, globally distributed marine toxins[END_REF].

Spirolides, as the other cyclic imines, are classified as "fast-acting" toxins because they induced rapid onset of neurological symptoms -including hyperactivity, jumping, piloerection, hyperextension of the back, stiffening and arching of the tail toward the head, tremors progressing to spams, as well as skeletal muscle paralysis and extension of the hind limbs, respiratory distress with marked abdominal breathing, tremors of the whole body and severe dyspnea -followed by death due to respiratory arrest within 3-50 minutes, whatever the route of administration is. It is worth noting that animals that did not die usually fully recovered with no detectable long-term effects, confirming that the Cyclic Imine toxins are rapidly detoxified and/or excreted (see above).

The LD 50 of some spirolide congeners, after i.p. injection to mice, revealed that these compounds are the most toxic among cyclic imines toxins. In particular, spirolide-C as well as, although contested (Munday et al., 2012;[START_REF] Otero | Pharmacokinetic and toxicological data of spirolides after oral and intraperitoneal administration[END_REF], 13-desmethyl spirolide-C and 20-methyl spirolide-G were the most toxic with LD 50 values as low as 0.007-0.008 mg/kg mouse, followed by 13,19-didesmethyl spirolide-C and spirolide-A (with LD 50 of 0.032 and 0.037 mg/kg mouse, respectively). The other spirolide congeners exhibit an apparently much lower toxicity (Table 2).

Interestingly, the comparison of the acute toxicity of spirolides and their chemical structure points out the importance of some structural requirements, such as the imine ring with the C31 methyl group, and the methyl group at C19, for the toxicological potency of the molecule. Although

showing similar rank orders in the acute toxicity response, the LD 50 of spirolide congeners after oral administration to mice via gavage are higher than those by i.p. injection, and even higher when administered by feeding (Table 2). The significant reduction in oral toxicity versus i.p.

administration of pure spirolide congeners is consistent with the absence of harmful effects in humans consuming shellfish contaminated with these cyclic imine toxins, with the exception of non-specific symptoms, such as gastric distress and tachycardia, reported in Canada [START_REF] Richard | Investigations into the toxicology and pharmacology of spirolides, a novel group of shellfish toxins, in Harmful Algal Blooms[END_REF]. However, the risk to human health of spirolide accumulation in shellfish is evidenced by the two following findings. First, the LD 50 of lipophilic shellfish extracts, containing mainly 13desmethyl spirolide-C, was shown to be higher after i.p. than oral administration to mice [START_REF] Richard | Investigations into the toxicology and pharmacology of spirolides, a novel group of shellfish toxins, in Harmful Algal Blooms[END_REF], and second, examination of the brains of mice and rats after i.p. injection with this spirolide congener revealed neuronal damage in the hippocampus and brain stem, with increased mRNA levels of muscarinic and nicotinic acetylcholine receptors [START_REF] Gill | Neural injury biomarkers of novel shellfish toxins, spirolides: A pilot study using immunochemical and transcriptional analysis[END_REF].

Table 2 near here -Molecular targets and mechanism of action of spirolide toxins

From the large number of compounds found in nature known to interact with nicotinic acetylcholine receptors (nAChRs), including a number of alkaloids and peptides from various origins, only a few are known to be produced by dinoflagellates. Dinoflagellate toxins known to interact with nAChRs include the spirolides, gymnodimines and pinnatoxins (for a recent review, see [START_REF] Stivala | Synthesis and biology of cyclic imine toxins, an emerging class of potent, globally distributed marine toxins[END_REF].

The nAChRs are ligand-gated ion channels mediating fast neurotransmission in the central and peripheral nervous systems (reviewed in [START_REF] Corringer | Nicotinic receptors at the amino acid level[END_REF][START_REF] Albuquerque | Mammalian nicotinic acetylcholine receptors: From structure to function[END_REF]. At the skeletal neuromuscular junction, the nAChR is a pentamer consisting of two α1-subunits, single β1and δ-subunits, and either a γ or a ε subunit in the embryonic or adult receptor-type, respectively (Fig. 3). The neuronal nAChRs in the peripheral and central nervous systems are composed of pentameric combinations of  and  subunits and exhibit a great heterogeneity in subtypes. To date, nine nAChR α subunits (α2-α10) and three nAChR β subunits (β2-β4) have been cloned from distinct animal species. In the peripheral nervous system, the primary subtype is the heteromeric α3β4 subtype, which is present in autonomic ganglia neurons. In addition, the 3 subunit may assemble with α5 and/ or β2 subunit to form a variety of nAChRs. The other major subtype of neuronal nAChR in the peripheral and central nervous system comprised 7 subunits forming a homomeric nAChR, as well as heteromers made up from the combination of different α subunits, or α and β subunits. These arrangements include the  nAChR, which is abundant and widely distributed in the central nervous system, and more complex subunit combinations with the presence of additional subunit types may exist. Thus, a major characteristic of neuronal nAChRs is their wide heterogeneity, based on both diverse stoichiometry and subunit composition.

GYMNODIMINES -Chemical characteristics of gymnodimine toxins

The gymnodimines present a 6-membered cyclic imine, bearing no methyl substituents, as part of the spiroimine ring system, an unsaturated lactone and a tetrahydrofuran ring (Fig. 5). Two groups can be distinguished according to the substitution pattern at the positions C17 and C18. The first gymnodimine group has a methyl substituent at C17 and an E-configured endocyclic double bond between C17 and C18. Gymnodimine-A and its 12-methyl derivative are part of this group. The second gymnodimine group contains gymnodimines B and C, which have an exocyclic double bond at C17 and a hydroxyl substituent at C18 with S and R stereochemistry, respectively.

-Gymnodimine producing organisms

The production of the gymodimines identified so far (gymnodimes-A, -B and -C) was exclusively attributed to the dinoflagellate Karenia selliformis [START_REF] Haywood | Comparative morphology and molecular phylogenetic analysis of three new species of the genus karenia (dinophyceae) from New Zealand[END_REF]) (formely known as Gymnodinium selliforme) in New Zealand and Australia (Table 3). However, the gymnodimine congener 12-methyl gymnodimine was reported as originating from another dinoflagellate in the United States, Alexandrium peruvianum (Van Wagoner et al., 2011) (Table 3), which was proven to also produce some spirolide congeners (see Table 1).

Table 3 near here -Toxicity of gymnodimine toxins

The gymnodimines are among the least toxic cyclic imines according to the mouse bioassay irrespective of the route of administration is (i.p. injection, gavage or feeding administration). Their acute oral toxicity, in particular that of gymnodimine-A, was shown to be between about 8-and more than 78-fold lower than the i.p. toxicity [START_REF] Munday | Acute toxicity of gymnodimine to mice[END_REF]) (Table 4). It is worth noting that gymnodimine-A was reported to be about 10-fold more toxic than gymnodimine-B after i.p. injection to animals. Remarkably, the LD 50 for gymnodimine-A was decreased by about 30-fold when administered to mice by intracerebroventricular administration, as compared to i.p. injection (i.e. 0.003 and 0.08-0.096 mg/kg mouse, respectively [START_REF] Kharrat | The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity[END_REF]) (Table 4). This indicates that gymnodimine-A preferentially affected the rodent central nervous system, further highlighting the risk of the Cyclic Imine toxins to human health may have when accumulated in shellfish at high concentrations.

Table 4 near here -Molecular targets and mechanism of action of gymnodimine toxins

Gymnodimine-A at nanomolar concentrations was reported to produce a concentration-dependent block of twitch tension responses evoked by nerve stimulation in isolated mouse phrenic-nerve hemidiaphragm-muscle preparations, without affecting muscle excitability and directly elicited muscle contraction. Also, gymnodimine-A, according to the nanomolar concentration used, either reduced the amplitude or completely blocked miniature-endplate potentials generated by the release of a single ACh quantum [START_REF] Kharrat | The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity[END_REF]. Similarly, gymnodimine-A blocked the amplitude of endplate potentials without affecting the resting membrane potential of muscle fibers. Such an action prevented endplate potentials to reach the threshold potential for action potential generation in muscles fibers, strongly suggesting that gymnodimine-A blocked endplate nAChRs. Direct evidence for such an action was obtained, using both the patch-clamp technique and iontophoretic ACh pulses of constant duration delivered by a micropipette on the surface of Xenopus skeletal myocytes expressing the embryonic muscle-type nAChR in their surface [START_REF] Kharrat | The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity[END_REF].

Gymnodimine-A was shown both to have no agonist action on such receptors, but to block reversibly the inward nicotinic currents elicited by the iontophoretic ACh pulses. Furthermore, the inhibition of ACh-activated currents by gymnodimine-A was clearly independent of the holding membrane potential, in other terms had no voltage-dependence. Interestingly, the neuromuscular block produced by gymnodimine-A could be reversed by continuous washout of the phycotoxin from the medium within 30 min. Also, the block of K + channels in motor nerve terminals by 3,4diaminopyridine (100 µM), which increases evoked quantal ACh release by nerve impulses (Van der Kloot and [START_REF] Van Der Kloot | Quantal acetylcholine release at the vertebrate neuromuscular junction[END_REF], was able to antagonize the action of the phycotoxin. In addition, gymnodimine-A, in a reversible manner, blocked ACh-evoked currents in Xenopus oocytes expressing heterologous human 7 nAChRs, and this block was independent of the holding membrane potential [START_REF] Kharrat | The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity[END_REF]. The X-ray crystal structure of gymnodimine-A in complex with Aplysia-AChBP has been determined [START_REF] Bourne | Structural determinants in phycotoxins and AChBP conferring high affinity binding and nicotinic AChR antagonism[END_REF]. The structures obtained indicate that gymnodimine-A was bound at the subunit interface, between loops C and F from opposing faces, in the same region where ACh binds. In addition, the structures highlighted the characteristic hydrogen bond between the iminium nitrogen in gymnodimine-A and the carbonyl oxygen of Trp 147 from loop C, and also the anchoring interactions of the tetrahydrofuran component in gymnodimine-A with the apical and membrane extremities of the binding site, along the subunit interface.

PINNATOXINS, PTERIATOXINS AND PORTIMINE -Chemical characteristics of pinnatoxins and pteriatoxins

To date, eight pinnatoxins (A-H) and three pteriatoxins (A-C) have been described. They all share the same common scaffold with a dimethyl substituted 7-membered cyclic imine as part of a spiroimine ring system, a 6,5,6-spiroketal ring system and a bridged ketal which is unique within the cyclic imine phycotoxins family (Fig. 6). The pinnatoxins and pteriatoxins are differentiated by the substituent at C33, which might be carboxyl (pinnatoxin-A), vinyl (pinnatoxins G and H), 5). Recently, a new pinnatoxin analog, pinnatoxin-H, was purified from a culture of the dinoflagellate Vulcanodinium rugosum from the South China Sea [START_REF] Selwood | Pinnatoxin H: a new pinnatoxin analogue from a South China Sea Vulcanodinium rugosum isolate[END_REF]. It is worth noting that the fatty acid esters of pinnatoxins A and G isolated from Canadian mussels were reported to likely originate from the shellfish than from the dinoflagellate (McCarron et al., 2012). Similarly, there is no conclusive evidence for the dinoflagellate source of pteriatoxins, isolated from the Japanese bivalve Pteria penguin [START_REF] Shumway | A review of the effects of algal blooms on shellfish and aquaculture[END_REF].

Table 5 near here -Toxicity of pinnatoxins and pteriatoxins

Acute toxicological studies with purified samples of pinnatoxin congeners, administered by i.p.

injection, revealed that pinnatoxin F was between about 2.7 and 5.1 times more potent than pinnatoxins E, G and H (Table 6), showing the following rank order of potency in the acute toxicity response: pinnatoxin F > pinnatoxin G > pinnatoxin E > pinnatoxin H (Munday et al., 2012b). A different rank order in the acute toxicity response (i.e. pinnatoxin F > pinnatoxin G > pinnatoxin H

> pinnatoxin E) was obtained for gavage administration. The oral toxicity of pinnatoxin-F was only between 5-and 6.5-fold higher than that of pinnatoxins G and H, but was about 93.3-fold higher than that of pinnatoxin E. As detailed previously for spirolide congeners and gymnodime-A, the oral toxicity of pinnatoxin-E was at least 49-fold lower than the i.p. toxicity, while comparatively smaller differences were observed for pinnatoxins F, G and H between the two routes of administration (Table 6). These findings are of great interest regarding the risk these toxins may have to human health. Only a few reports were concerned with the acute toxicity of pteriatoxins, but they revealed that these toxins produce animal lethality at doses between 0.008 and 0.1 mg/kg when administrated to mouse by i.p. injection, with toxic symptoms resembling those of pinnatoxins (see [START_REF] Takada | Structural determination of pteriatoxins A, B and C, extremely potent toxins from the bivalve Pteria penguin[END_REF]. The atypically stability of the aliphatic cyclic imine in pinnatoxins [START_REF] Jackson | Stability of cyclic imine toxins: interconversion of pinnatoxin amino ketone and pinnatoxin A in aqueous media[END_REF] is probably associated to their high oral toxicity, highest among the Cyclic Imine toxins.

-Molecular targets and mechanism of action of pinnatoxins and pteriatoxins

The use of successful strategies for the chemical synthesis of pinnatoxins A and G [START_REF] Beaumont | Marine Toxins with Spiroimine Rings: Total Synthesis of Pinnatoxin A[END_REF][START_REF] Stivala | Synthesis and biology of cyclic imine toxins, an emerging class of potent, globally distributed marine toxins[END_REF] allowed the production of substantial amounts of these toxins in pure form which permitted performing electrophysiological and functional studies, as well as competition binding studies together with computational molecular modeling, leading to the revision of the mode of action previously proposed for pinnatoxins. Thus, based on such analyses it has been unequivocally established that pinnatoxins A and G are potent antagonists of nAChRs [START_REF] Aráoz | Total synthesis of pinnatoxins A and G and revision of the mode of action of pinnatoxin A[END_REF]. The rank order of potency for pinnatoxin-A on nAChR subtypes determined by functional analyses was the following: human 7 > Torpedo 2 β1γδ > human 4β2. The reversibility of the pinnatoxin-A activity was faster for the  nAChR, while it was slowly reversible for the 7 nAChR subtype. Interestingly, pinnatoxin-A exhibited higher affinity than pinnatoxin-G for the human neuronal α7 nAChR subtype. In contrast, a synthetic aminoketone derivative of pinnatoxin-A with an open imine ring showed no activity on the various nAChR subtypes studied, a feature supporting the central role of the seven-membered cyclic imine ring for the potent antagonism of this family of toxins [START_REF] Aráoz | Total synthesis of pinnatoxins A and G and revision of the mode of action of pinnatoxin A[END_REF].

Also, pinnatoxins E, F and G were reported to produce concentration-dependent reductions in nerve-evoked twitch tension in isolated rat hemidiaphragm preparations, with a rank order of potency of F > G > E, while no effects were detected on directly muscle-evoked twitch responses.

Pinnatoxins F and G moreover reduced the amplitude of spontaneous miniature endplate potentials and evoked endplate potentials at skeletal neuromuscular junctions, without affecting miniature endplate potential frequency or the resting membrane potential of the muscle fibers. These results

show that pinnatoxins E, F and G are potent neuromuscular blockers and cause toxicity by acting as antagonists at muscle-type nicotinic acetylcholine receptors [START_REF] Hellyer | Neuromuscular blocking activity of pinnatoxins E, F and G[END_REF].

Further ligand-binding studies were performed to examine whether pinnatoxin-A had any action on other neurotransmitter receptors, including those coupled to G-proteins, and neurotransmitter transporters. However, no significant action, or very little action, was detected using a high concentration of pinnatoxin-A (10 µM) on the different receptors studied including: adrenergic receptors (Alpha1A, Alpha1B, Alpha1D, Alpha2A, Alpha2B, Alpha2C, Beta1, Beta2, Beta3) ;

PROROCENTROLIDES AND SPIRO-PROROCENTRIMINE -Chemical characteristics of prorocentrolide and spiro-prorocentrimine toxins

Prorocentrolides are the only members of the cyclic imine phycotoxins family that do not contain a spiroimine group. Their chemical structure includes a 26-membered carbo-macrocycle and a 28membered macrocyclic lactone arranged around a 3,4,4a,5,8,8a-hexahydroisoquinoline that incorporates the characteristic cyclic imine group. Six prorocentrolides have been reported to date (Fig. 7), which differ by the substituents at the positions 4 (hydrogen, hydroxy or sulfonate), 9

(methyl or exocyclic vinyl), 14 (hydroxy or acetate ester), 29 (methyl or hydrogen) and 30 (hydroxy or sulfonate). Prorocentrolide-B shows specific structural modifications at the positions 10 (exocyclic vinyl), 15 (hydrogen instead of hydroxy), 16 (hydroxy instead of hydrogen) and 29

(unsubstituted endocyclic E double bond).

A single spiro-prorocentrimine compound is currently known (see Fig. 1), whose chemical structure contains a 25-membered carbo-macrocycle and a 15-membered macrocyclic lactone arranged around a 6,6-spiroimine ring system. The overall structure and the stereochemistry of substituents have been unambiguously determined by X-ray crystallography [START_REF] Lu | Spiro-prorocentrimine, a novel macrocyclic lactone from a benthic Prorocentrum sp. of Taiwan[END_REF].

-Prorocentrolide and spiro-prorocentrimine producing organisms

To date, the production of prorocentrolide congeners and spiro-prorocentrimine is well-known to be restricted to primarily benthic or epiphytic Prorocentrum sp. which, unlike their pelagic counterparts, does not form dense blooms. Hence, prorocentrolide A was isolated from Prorocentrum lima in Japan and Taiwan, and the structurally related cyclic imine prorocentolide B from Prorocentrum maculosum in tropical waters. The Taiwanese Prorocentrum lima also produces two prorocentrolide analogs, 4-hydroxy and 14-O-acetyl-4-hydroxy prorocentrolides. Finally, spiroprorocentrimine was isolated along with prorocentrolide A from another Taiwanese, still unidentified, strain of Prorocentrum sp. (Table 7).

Table 7 near here -Toxicity of prorocentrolide and spiro-prorocentrimine toxins

Although only a few reports were concerned with the acute toxicity of prorocentrolide and spiroprorocentrimine toxins, they revealed that, when administrated to mouse by i.p. injection, prorocentrolide A had an animal lethality of 0.4 mg/kg mouse [START_REF] Torigoe | Prorocentrolide, a toxic nitrogenous macrocycle from a marine dinoflagellate, Prorocentrum lima[END_REF], while spiro- prorocentrimine exhibited much less toxicity (i.e. animal lethality occurred at 2.5 mg/kg mouse) than other known marine Cyclic Imine toxins [START_REF] Lu | Spiro-prorocentrimine, a novel macrocyclic lactone from a benthic Prorocentrum sp. of Taiwan[END_REF].

-Molecular targets and mechanism of action of prorocentrolide and spiro-prorocentrimine toxins

The molecular targets for these Cyclic Imine toxins have not yet been determined, because of the lack of purified or synthetic toxins.

CONCLUSION

In conclusion, this short review shows that the well-chemically characterized natural products derived from some toxic dinoflagellates species constitute a novel source of potent nAChR-ligands molecules. The distinguishing feature of these phycotoxins is the presence of a cyclic imine moiety in their structure, which constitutes a chemical signature associated with their capacity to interact with muscle-types and neuronal-nAChR subtypes. Thus, spirolides, gymnodimines and pinnatoxins, which are recognized "fast-acting toxins", are known to broadly target muscle-and neuronal-type nAChRs, generally with high affinity for the various receptor subtypes studied. The sites of interaction of these toxins on nAChRs include amino acid residues highly conserved in the sequences of muscle-and neuronal-type receptors. Another important aspect in the action of these phycotoxins is the lack of animal species-selectivity, since similar actions have been reported on frog, fish, chick, rodent, and human nAChRs. The toxicological profile reported for these phycotoxins, during rodent bioassays, is mostly due to their specific interaction with the nAChRs.

The competitive binding to nAChRs has been exploited to design and develop new tests to detect spirolides, gymnodimines and pinnatoxins in contaminated shellfish with better accuracy than the broad spectrum mouse bioassay. These tests are important in the food safety field, since shellfish constitutes a worldwide rich food resource that may be contaminated by toxins produced by harmful dinoflagellates. Additional appropriate toxicological studies must be developed to assess the risk these phycotoxins may have to human health, since at present there is no strong evidence linking the presence of spirolides, gymnodimines and pinnatoxins in shellfish tissues with human intoxications. Also, more data is needed on the risks of chronic exposure to these phycotoxins. Shellfish frequently contains varying amounts of these toxins, at low or high concentrations, which is a risk factor for human health that cannot be neglected. Furthermore, shellfish concentrate these toxins in their edible tissues, and can act as vector for transferring these phycotoxins through the marine food web, thus menacing wildlife.

Figure 1 near hereFigure 1 .

 1 Figure 1 near here

Figure 2

 2 Figure 2 near here

Figure 2 .

 2 Figure 2. Chemical structures of the known spirolide toxins.

Figure 3

 3 Figure 3 near here

Figure 3 .

 3 Figure 3. Three-dimensional structure of the muscle-type nAChR (Torpedo marmorata) obtained by cryoelectron microscopy (Protein Data Bank code 2BG9). The five subunits are colored in red, yellow, green, cyan and blue. The two antagonist sites where the spiroimine toxins can bind are shown as magenta-colored surfaces. Left: lateral view showing the extracellular (ECD), transmembrane (TMD) and intracellular (ICD) domains. Right: top view showing the organization of the five subunits arranged around the central pore.

Figure 4

 4 Figure 4 near here

Figure 4 .

 4 Figure 4. Experimental approaches used to study the action of spirolides and other spiroimine phycotoxins using electrophysiological techniques. (A) Mature female Xenopus laevis frog. (B) Oocytes at stages V to VI of development, surgically removed from an anesthetized mature female X. laevis under anesthesia. (C) Western blot of Torpedo marmorata purified membranes showing the g and d subunits of the nAChR and the rapsyn ( r ) protein. (D) Manually defolliculated oocytes in which one is microinjected with 50 nL of purified Torpedo membranes using a Nanoliter 2010 Micro 4 Controller injector mounted on a microscope. (E, F) Staining of nAChRs incorporated to the membrane of an oocyte using fluorescent α-Bungarotoxin, Alexa Fluor® 488 conjugate. (G) Inward nicotinic current triggered by an ACh pulse of 15 seconds duration, recorded in an oocyte micro-injected 4 days previously with purified Torpedo membranes. Perfusion of 13-desmethyl spirolide C (13-SPX-C) had no agonist action (blue tracing above recording), but complete blocked the ACh-evoked current

Figure 5 near hereFigure 5 .

 5 Figure 5 near here

Further insight into the

  interaction between gymnodimine-A and the muscle-type    nAChR was gained in competition binding studies using [ 125 I]-bungarotoxin as a tracer and HEK-293 cells expressing this nAChR. Gymnodimine-A totally displaced, in a concentration-dependent manner, [ 125 I]-bungarotoxin from HEK-293 cells expressing the muscle-type   nAChR or the chimeric neuronal 7-5-HT3 nAChR. Furthermore, gymnodimine-A inhibited the specific [ 3 H]epibatidine binding on HEK293 cells expressing either  or  neuronal nAChRs. These data are consistent with the fact that gymnodimine-A was highly toxic when applied directly into the mouse central nervous system by intracerebroventricular injection. The rank order of potency for gymnodimine-A on the various nAChRs studied was: 7-5HT3 > 1 2  >  > [START_REF] Kharrat | The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity[END_REF]. In another study the rank order for binding affinity (Ki) was α7, α6β3β4α5 > rat α3β4 > human α3β4, α4β4 > rat α4β2, human α4β2[START_REF] Hauser | Comparison of acetylcholine receptor interactions of the marine toxins, 13-desmethylspirolide C and gymnodimine[END_REF]. Some 6,6-spiroimine analogs of gymnodimine-A have been chemically synthesized, and were shown to block ACh-evoked nicotinic currents in Xenopus oocytes having incorporated the Torpedo muscle-type (  ) nAChR in their membrane, or expressing the human nAChR[START_REF] Duroure | 6,6-Spiroimine analogs of (-)-gymnodimine A : synthesis and biological evaluation on nicotinic acetylcholine receptors[END_REF]. Although these spiroimine analogs are about 40-45 times less active than gymnodimine-A, the fact that they block both types of nAChRs indicate that the 6,6-spiroimine moiety is a key structural factor required for blocking nAChRs in the gymnodimine family.

  glycinyl (pinnatoxins B and C), butyrolactone (pinnatoxin-F) or hydroxy/keto-butyric acid (pinnatoxins D and E), as well as S-cysteinyl derivatives (pteriatoxins A-C). Other positions with substituent variability are 21, 22 and 28, for which we observe two substitution patterns: H, H and OH (pinnatoxins A, B, C, G and pteriatoxins A, B, C), and CH 3 , OH and H (pinnatoxins D, E, F, H), respectively.

Figure 6 near hereFigure 6 .

 6 Figure 6 near here

  muscarinic receptors (M1, M2, M3, M4 and M5); serotoninergic receptors (5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT5A, 5-HT6, 5-HT7); GABAA receptor, dopaminergic receptors (D1, D2, D3, D4 and D5), Histamine receptors (H1 and H2), Opioid (DOR), Opioid (KOR), µ-Opioid (MOR) and the dopamine transporter (DAT), norepinephrine transporter (NET) and serotonin transporter (SERT). Furthermore, no activity on Ca V 1.1 calcium channels (type-L) originally proposed to be the target of pinnatoxins, was observed[START_REF] Aráoz | Total synthesis of pinnatoxins A and G and revision of the mode of action of pinnatoxin A[END_REF]. These experimental results reveal an extraordinary selectivity of pinnatoxin-A for the nAChRs.The evidence that spirolides, gymnodimines and pinnatoxins target nAChRs prompted the development of target-directed functional method, based on the mechanism of action of competitive antagonists on nAChRs. Thus, several non-radioactive ligand-binding methods have been developed(Vilariño et al., 2009; Fonfría et al., 2010; Otero et al., 2011; Aráoz et al., 2012; Rodriguez et al., 2013) to replace the traditional mouse bioassay which has numerous drawbacks related to sensitivity and specificity, apart from the ethical concern of using large number of animals, as recently discussed (Daneshian et al., 2013).-PortimineA new Cyclic Imine toxin, named portimine, has been recently discovered from the pinnatoxin producing dinoflagellate Vulcanodinium rugosus from Northland (New Zealand)[START_REF] Selwood | Portimine: A bioactive metabolite from the benthic dinoflagellate Vulcanodinium rugosum[END_REF]. The structure of portimine, including the relative configurations, was elucidated by spectroscopic analyses. The cyclic imine moiety consists of an unprecedented five-membered ring with a spiro-link to a cyclohexene ring (see Fig.1). Portimine contains only 14 carbons in its macrocycle, making it the smallest Cyclic Imine toxin isolate to date. Such structural factors have a large impact on the toxin toxicity, since it has a much lower toxicity in rodent bioassays after i.p. administration (LD 50 = 1.57 mg/kg mouse) than any other Cyclic Imine toxin. In contrast, portimine exhibits high toxicity in vitro to mammalian cells, with an LC 50 of 2.7 nM to mouse leukemia P388 cells, and activation of caspases indicates that it has apoptotic activity.

Figure 7 near hereFigure 7 .

 7 Figure 7 near here

Table 1 .

 1 Dinoflagellate species known to produce spirolides, their location and produced compounds.

		Canada	Spirolides A, B, C, C2, C3, D, D2, D3, H and I 13-desmethyl Spirolides C and D
		Norway	20-methyl Spirolide G
			Spirolides D and G
		Denmark	13-desmethyl Spirolides C and D
			13,19-didesmethyl Spirolide C
			Spirolide D
	Alexandrium ostenfeldii	Italy	13-desmethyl Spirolides C and D 13,19-didesmethyl Spirolide C 27-hydroxy-13,19-didesmethyl Spirolide C
			27-oxo-13,19-didesmethyl Spirolide C
			27-hydroxy-13-desmethyl Spirolide C
		Scotland	Spirolide C
			Spirolides C and D
		Ireland	13-desmethyl Spirolide C
			20-methyl Spirolide G
		Mediterranean Sea	Spirolides B, C and D 13-desmethyl Spirolides C and D
	Alexandrium peruvianum	Ireland	13-desmethyl Spirolides C and D
		Unired States	Spirolide D 13-desmethyl Spirolides C and D

Table 2 .

 2 The 50% lethal doses (LD 50 ) of spirolide congeners according to their administration route.

	Spirolide	Administration mode	LD 50 (mg/kg mouse)	Ratio (compared to i.p.)
		Intraperitoneal injection	0.037	1
	Spirolide A	Gavage	0.24-0.55	6.5-14.9
		Feeding	1.2-1.3	32.4-35.1
	Spirolide B	Intraperitoneal injection Gavage	0.099 0.440	1 4.4
		Intraperitoneal injection	0.008	1
	Spirolide C	Gavage	0.053-0.18	6.6-22.5
		Feeding	0.5-0.78	62.5-97.5
		Intraperitoneal injection	0.007-0.028	1
	13-desmethyl Spirolide C	Gavage	0.13-0.16	4.6-22.9
		Feeding	0.5-1	1 7 . 9 -142.9
	13,19-didesmethyl Spirolide C	Intraperitoneal injection	0.032	1
	27-hydroxy-13-desmethyl Spirolide C	Intraperitoneal injection	>0.027	1
	27-oxo-13,19-didesmethyl Spirolide C	Intraperitoneal injection	>0.035	1
	Spirolide E	Intraperitoneal injection	>1.000	1
	Spirolide F	Intraperitoneal injection	>1.000	1
		Intraperitoneal injection	0.008->0.063	1
	20-methyl Spirolide G	Gavage	0.088-0.16	<1.4
		Feeding	0.5-0.63	<7.9

Table 3 .

 3 Dinoflagellate species known to produce gymnodimine congeners.

	Karenia selliformis	New Zealand Australia	Gymnodimines A, B and C Gymnodimine A
	Alexandrium peruvianum Unired States	12-methyl Gymnodimine

Administration mode LD 50 (mg/kg mouse) Ratio (compared to i.p.)

	Gymnodimine A	Intraperitoneal injection	0.08-0.096	1
		Intracerebroventricular injection	0.003	0.03-0.038
		Gavage	0.755	7.9-9.4
		Feeding	4.057->7.5	42.3->78.1
	Gymnodimine B	Intraperitoneal injection	0.800	1

Table 4 .

 4 The 50% lethal doses (LD 50 ) of gymnodimine congeners according to their administration route.

Table 6 near here

 6

	Vulcanodinium rugosum	Australia	Pinnatoxins A, E, F and G
		New Zealand	Pinnatoxins E and F
		Japan	Pinnatoxin G
		France	Pinnatoxin G
		China	Pinnatoxin H

Table 5 .

 5 Dinoflagellate species known to produce pinnatoxin congeners.

	Pinnatoxin	Administration mode	LD 50 (mg/kg mouse)	Ratio (compared to i.p.)
		Intraperitoneal injection	0.045-0.057	1
		Gavage	2.800	49.1-62.2
		Intraperitoneal injection	0.013-0.016	1
		Gavage	0.025-0.03	1.6-2.3
		Feeding	0.05-0.077	3.1-5.9
		Intraperitoneal injection	0.043-0.05	1
		Gavage	0.150	3-3.5
		Feeding	0.400	8-9.3
		Intraperitoneal injection	0.067	1
		Gavage	0.163	2.4

Table 6 .

 6 The 50% lethal doses (LD 50 ) of pinnatoxin congeners according to their administration route.

	Pinnatoxin E
	Pinnatoxin F
	Pinnatoxin G
	Pinnatoxin H

Table 7 .

 7 Dinoflagellate species known to produce prorocentrolide congeners and spiroprorocentrimine.

		Japan	Prorocentrolide A
	Prorocentrum lima	Taiwan	Prorocentrolide A 4-hydroxy Prorocentrolide
			14-O -acetyl-4-hydroxy Prorocentrolide
	Prorocentrum maculosum Tropical waters	Prorocentrolide B
	Prorocentrum sp.	Taiwan	Prorocentrolide A Spiro-prorocentrimine

Acknowledgements

Work performed in the laboratory of authors was funded by the "Agence Nationale de la Recherche" (France, grant AQUANEUROTOX ANR-12-ASTR-0037-1), and by the National Institutes of Health (USA, grant NIGMS R01 GM077379 to A.Z).


Lise Gross

Édouard Lhomme

Chloé Pasin

Laura Richert

Rodolphe Thiébaut

Edouard Lhomme

Rodolphe Thiebaut
email: rodolphe.thiebaut@u-bordeaux.fr

Ebola vaccine development: Systematic review of pre-clinical and clinical studies, and meta-analysis of determinants of antibody response variability after vaccination

Keywords:

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Following the deadly 2013-2016 epidemic in West Africa, there has been an accelerated development of several candidates for an Ebola preventive vaccine. Outbreaks of Ebola virus disease (EVD) have occurred recurrently and unpredictably for the past 40 years with a high lethality rate [START_REF] Liu | Ebola virus disease: from epidemiology to prophylaxis[END_REF]. The 2013-2015 outbreak was unprecedented in scale, with over 28,000 cases and more than 11,000 deaths (Ebola Situation Report, 2016). Incidental cases are still reported as recently in the Democratic Republic of Congo in May 2017 [START_REF] Dhama | Ebola from emergence to epidemic: the virus and the disease, global preparedness and perspectives[END_REF]. In the absence of any specific treatment, EVD prevention and control measures are primarily based on case identification and isolation, early non-specific medical care, surveillance of suspect cases, and safe burial practices [START_REF] Henao-Restrepo | Efficacy and effectiveness of an rVSV-vectored vaccine in preventing Ebola virus disease: final results from the Guinea ring vaccination, open-label, clusterrandomised trial (Ebola Ça Suffit!)[END_REF]. These measures are now sometimes complemented by ring vaccination of contacts of cases, based on the promising results of a phase III cluster-randomized ring vaccination efficacy trial conducted in Guinea in 2015 [START_REF] Ohimain | Recent advances in the development of vaccines for Ebola virus disease[END_REF]. However, the vaccine used for ring vaccination (rVSV ZEBOV vaccine) is not yet licenced and conducting new efficacy trials for licencing is not feasible in the absence of a large outbreak. Nevertheless, preparation for future outbreaks is required and the licensing of one or several preventive vaccines for stockpiling is a priority.

Several candidate vaccines strategies have been investigated since the first reported EVD outbreak in 1976. During and following the 2013-2015 epidemic, the process of vaccine development has been substantially accelerated, and several strategies have been moved into clinical phases. Despite the promising results of the ring vaccination trial in Guinea [START_REF] Ohimain | Recent advances in the development of vaccines for Ebola virus disease[END_REF], many questions, such as durability of immune responses, and immune responses and protection in specific sub-groups such as young children, remain to be addressed and Ebola vaccine development continues to be very active. Based on their delivery technologies, several candidate vaccine platforms can be distinguished: whole-virus vaccines, DNA vaccines, virus-like particles vaccines, and recombinant vaccines with different viral vectors (vesicular stomatitis virus or VSV, modified vaccinia Ankara or MVA, human adenovirus or Ad, and chimpanzee adenovirus or ChAd) (World Health Organisation, 2013). Each platform may use specific dose levels and Ebola antigen inserts.

Vaccine trials aim to assess vaccine safety and immunogenicity in phase I and II trials in humans prior to testing for a protective effect in phase III. Assessment of vaccine efficacy during preclinical and clinical studies is required to go through the vaccine license steps. Clinical protection from EVD in human populations is impossible to observe outside an epidemic period. In the nonepidemic context, Ebola vaccines are thus currently evaluated by using a main immunogenicity endpoint: the antibody response after vaccination. There is no definite evidence that antibody response is the correlate of protection or surrogate endpoint for efficacy in humans, that is a specific immune response to vaccine associated with vaccine-induced protection [START_REF] Sullivan | Correlates of protective immunity for Ebola vaccines: implications for regulatory approval by the animal rule[END_REF] and it may vary according to the vaccine platforms (Sullivan et al., 2000a,b). However, we know that antibody response is correlated with survival after challenge in nonhuman primate models, which is the nearest model to humans for EVD and hence the animal gold standard to test candidate Ebola vaccines; this association is found consistently for different Ebola candidate vaccines [START_REF] Wong | Immune parameters correlate with protection against ebola virus infection in rodents and nonhuman primates[END_REF]Food and Drug Administration, 2015;[START_REF] Sridhar | Clinical development of Ebola vaccines[END_REF].

For these reasons, antibody response is used as the main criterion to assess the Ebola candidate vaccines in phase I/II trials. In the absence of the possibility to conduct additional phase III trials, regulatory pathways not requiring such efficacy results are also under discussion (Food and Drug Administration, 2015). Significant variations in antibody responses are observable across studies, which could be due to the different types of vaccines evaluated, or not. Various factors are suspected to influence the level of antibody response beyond the vaccine features (vaccine platform, Ebola viral insert, dosage, single injection or boost, . . . ) such as the measurement techniques (time of measurement, antigen used to detect antibody response, . . . ) or the population type (human or nonhuman primates, age, sex, study site, . . . ). There is a lack of quantification of the contribution of each factor in the observed variation of the reported antibody responses.

Although previous reviews exist on Ebola vaccines [START_REF] Ohimain | Recent advances in the development of vaccines for Ebola virus disease[END_REF][START_REF] Sridhar | Clinical development of Ebola vaccines[END_REF][START_REF] Wu | Ebolavirus vaccines: progress in the fight against Ebola virus disease[END_REF], the specific topic of antibody response determinants has not yet been addressed by a systematic review or meta-analysis. Yet, the identification of factors potentially associated with antibody response after Ebola vaccination could provide relevant information for further vaccine trials and for regulatory decision making.

By conducting this systematic review with a meta-analysis, we aimed to determine whether the reported antibody response variability in Ebola vaccine trials is not only determined by the vaccine platform but also by other characteristics of vaccine and by population and measurement characteristics and to quantify these factors.

Methods

Search strategy and selection criteria

Studies were identified by searching electronic databases PubMed and Scopus. Pubmed was searched using the following terms: (« hemorrhagic fever, ebola » [MeSH Terms] OR « ebola » [All fields] OR « ebolavirus » [MeSH Terms] OR « ebolavirus » [All fields]) AND (« vaccines » [MeSH Terms] OR « vaccines » [All fields] OR « vaccine » [All Fields]). Scopus was searched using the following terms TITLE-ABS-KEY (ebola) AND TITLE-ABS-KEY (vaccine). Additionally, the Clinicaltrials.gov website was searched to identify unpublished and ongoing studies. Several experts in the field were contacted to find papers which could be not indexed in databases. Reference lists of relevant papers and reviews were examined to identify further articles.

The search was performed on March 23, 2016 and updated as of February 24, 2018 with a publication date limit of the same date in order to identify all published studies which met the inclusion criteria and without restriction on language. All preventive Ebola vaccine clinical trials conducted in humans or in nonhuman primates and with a measure of Ebola Zaire antibody titre after vaccination were included in our systematic review. Studies were excluded in case of duplicate study, studies without original data, preclinical studies conducted in animals other than nonhuman primates or in vitro experimentation.

Data extraction

A first step of selection was performed on the title and abstract, and then a second step was performed after reading the full article. Two authors independently assessed each full article to include papers matching the review's inclusion criteria. Disagreements between reviewers were resolved by consensus.

Data were extracted by two independent reviewers, with differences reconciled by consensus. The following variables were extracted: paper identification (title, first author, publication year), study design, inclusion and exclusion criteria, characteristics of the population (number of subjects; human or nonhuman primates; proportion of women, average age and study site for clinical trials; and animal species for pre-clinical studies using nonhuman primates), characteristics of vaccine (vaccine platform in terms of delivery technology used, specific vector for recombinant vaccines, Ebola viral insert, dosage, route of administration, vaccination schedule), characteristics of measurement techniques (time interval between last injection and measure, strain and nature of antigen used to detect antibody response, measurement method), antibody response after vaccination (geometric mean titre and its variance). Regarding the antibody response after vaccination, geometric mean titre was extracted from the text or estimated from figures. If a single vaccination group had more than one measure of antibody response, data from measurement after each injection were extracted. Therefore, if available, measurement post-prime and measurement post-boost from a same vaccination group were both included in our meta-analysis. If several measurements post-prime or if several measurements post-boost were available, for each injection we extracted the one closest to 28 days after injection, which is a standard time point in Ebola vaccine trials. Variance of titre (within-group variance) was extracted directly from the text or calculated from confidence interval or from individual values. The present study was registered in PROSPERO (no. 54303).

Data analysis

For all analyses, the statistical unit used was the vaccination group (one or several groups for a single study), i.e. a protocoldefined group undergoing the same intervention and follow-up procedures (such as a randomized arm of a clinical trial or an animal group in NHP studies).

First descriptive analyses were performed among all groups, separately for nonhuman primates and for humans. Then, a random-effect meta-regression analysis was performed including only human groups with 8 individuals or more. This threshold allowed both to have sufficient inter-individual variability in each group and to avoid excluding too many groups. Thus, it was not possible to perform the regression analysis with NHP studies because of the usual small sample size of the groups. The effect of every potential determinant of antibody response was assessed through fixed effects. A random intercept was allowed to capture between-group variability not explained by the fixed effects. The residual variance (within-group variance) was fixed in the model according to the values resulting from data extraction as described by [START_REF] Van Houwelingen | Advanced methods in meta-analysis: multivariate approach and meta-regression[END_REF].

Each potential determinant associated in unadjusted analyses with a p-value <0.25 was included in the multivariable model using forward step-wise selection. The heterogeneity was checked visually with forest plots and quantified by using the Q test. The proportion of total variation across groups due to heterogeneity (I 2 ) and the amount of variability explained by the factors included in the random-effect model (R 2 ) were estimated. Antibody titres after vaccination were log transformed in the model.

For the meta-regression analysis, the dosage variable was categorized into "low dose" or "high dose" per vaccine platform, since units of measurement for dose level were platformdependent. For each unit of dose measurement and each vaccine platform, the average dose level among the human groups included in the meta-regression model was used as a classification threshold for this variable; if only one dose level was assessed for a vaccine platform, the dosage variable was defined as undifferentiated. The absence of interaction between vaccine platform and dosage was checked (likelihood ratio test: p = 0.223).

All analyses were performed using the metafor package of R (i386 3.2.2 version, the R Foundation, Vienna, Austria).

Results

Study selection

The selection process of the studies and vaccination groups is described in Figure 1.

The search yielded a total of 2166 studies. Of these, 49 met the inclusion criteria to the research question corresponding to 202 vaccination groups. Unpublished clinical trials and one trial found by contact with an expert were excluded since no results were available. Studies not reporting any antibody measurements were also excluded. This led to the exclusion of the "Ebola ça suffit" ring vaccination trial conducted in Guinea, the only trial that was able to assess clinical efficacy in humans so far [START_REF] Ohimain | Recent advances in the development of vaccines for Ebola virus disease[END_REF]. This trial was conducted under emergency conditions and did not collect blood samples for immunogenicity measurements.

Table 1 shows details of all trials included in the systematic review: 32 studies were conducted in NHP, 13 trials in humans were phase 1, two trials phase 1/2, and two phase 2. The number of trials has increased significantly since the last outbreak of EVD. Clinical trials were conducted mostly in Europe and North America (Figure 2).

Description of included vaccination groups

Among the 202 vaccination groups included in our systematic review, 74 were human groups and 128 were non-human primate groups. The distribution of the number of individuals by groups is presented in Figure 3. The vast majority (82.4%) of human groups included 8 or more individuals, while only 6 for non-human primate groups (range 2; 22 with an average of 4.1 individuals by group). Characteristics of nonhuman primate and human groups are described in Tables 2 and3, respectively.

There is a wide heterogeneity of features among studies included in the systematic review. Vaccine platforms varied between studies, especially in NHP (18 different vaccine platforms in NHP groups versus 8 in human groups). The strain of Ebola virus used as vaccine insert or for the antibody detection after vaccination was also variable. For almost a third of the human groups, the detection of antibody response was done with a heterologous strain. The time interval between the last vaccination and the antibody detection was also remarkably variable (range 3; 371 days). Among all the 202 vaccination groups, the mean antibody titre ranged from 0 (for a group of NHP infected by the simian/human immunodeficiency virus prior to the Ebola vaccination) to 5.81 log10, with an average of 2.97 (95% CI: [2.84; 3.10]).

The NHP groups had a crude antibody response level that was significantly higher than the human groups (p = 0.006): in NHP groups the log10 geometric mean titre ranged from 0 to 5.81 with an average of 3.10 (95% CI: [2.93; 3.27]), and in human groups the titre ranged from 0.90 to 4.60 with an average of 2Á75 (95% CI: [2.57; 2.93]) Figure S1 (appendix) shows antibody responses in human groups and in NHP groups.

Meta-regression of factors associated with variability in antibody response levels in humans and evaluation of between-groups heterogeneity

Sixty-one human vaccination groups with 8 individuals or more were included in the meta-regression analysis.

Among these, 32 were vaccinated with a low dose of vaccine, 19 with a high dose (for 10 groups, the dose category was undeterminable as only one dose level was assessed for the given vaccine platform).

The distribution of the antibody titres after Ebola vaccination per vaccination group is shown by vaccine platform in Figure 4. The antibody response seems to be higher in groups with a primeboost strategy (Ad26/MVA or ChAd3/MVA) than in the other groups. The distribution of the antibody titres by viral strain used for antibody detection is presented in Figure S2 (appendix).

In univariate meta-regression analyses (appendix: Table S1), the antibody response after Ebola vaccination was significantly associated with the vaccine platform (p < 0.001), the viral strain used to detect the antibody response after vaccination (p < 0.001), the year of publication (for publication in 2014 and after versus before 2014: +1.15, p < 0.001), the mean age of vaccinated population (for !39 years versus <32 years: +0.90; p < 0.001), the vaccine dosage (for high dose versus low dose: +0.57, p = 0.006), the use of a vaccine boost (for boost versus no boost: +0.63, p = 0.009), the similarity between the viral strain used as vaccine insert and the viral strain used to detect the antibody response (for identical strains versus different strains: À0.74, p = 0.009), the site of the study (p = 0.014), the time interval between the last vaccine injection and the antibody measure (for <28 days versus !28 days: +0.70, p = 0.021), and the Ebola species of vaccine insert (for multivalent and other species versus monovalent Zaire: À0.47, p = 0.027). Alone, the vaccine platform was the factor which explained the largest part of heterogeneity among all the studied factors (R 2 for vaccine platform = 55%). For all the univariate models, the heterogeneity was very high with I 2 ranging from 97% to 99%.

Results of the final multivariate meta-regression model are shown in Table 4. High heterogeneity was found with a I 2 of 95% and a R 2 of 68%, even after adjustment on the factors associated with antibody response in this final model. This emphasises the lack of factors that explained the antibody response among the variables included in the model. Vaccine platform and viral strain used for detection were the two factors which were independently associated with antibody response after vaccination against Ebola. Compared to the MVA vaccine platform, the recombinant vaccines using DNA or Ad26 (associated or not with an injection of MVA vaccine), ChAd3or VSV vectors were significantly associated with a higher antibody response after vaccination (more than 1.2 log10 units more compared to MVA alone). The statistical association between the vaccine platform and the antibody response was strong and consistent regardless of which other variables were included in the model (sensitivity analyses, data not shown). The antibody response using Makona strain for antibody detection was significantly higher than with use of Mayinga strain (1 log10 unit more compared to the Mayinga strain). By contrast, the antibody response with Kikwit detection strain was not significantly different from the ones with Mayinga strain.

The vaccine dosage, analysed as a binary variable of high versus low dose in the present analyses, was not found to be associated with antibody response variability. Different classifications were tested for this variable (same threshold across the different vaccine platforms corresponding to the mean dose level for categorizing into "low-dose" and "high-dose" groups, classification into three categories, classification of groups with undifferentiated dosages into "low-dose" or into "high-dose" groups), but the dosage was never significant in the multivariate models in these sensitivity analyses (data not shown), nor was the interaction between dose and vaccine platform.

In additional sensitivity analyses, a full model including all variables significantly associated with the antibody response in univariate models (i.e. with no forward selection procedure) did not modify heterogeneity (I 2 = 92%) compared to the model presented above. In the full model, the vaccine platform was significantly associated with the antibody response (p = 0.002), but the viral strain used to detect the antibody response after vaccination was not (p = 0.996). The other variables were not associated with the antibody response.

Discussion

This systematic review on preventive Ebola vaccine trials has found 49 studies conducted in humans or in NHP. The metaanalysis, using a random-effect inverse variance meta-regression including 61 human vaccination groups, showed a major part of antibody response variability in humans that remained unexplained by the factors included in the model. Indeed, the betweengroup heterogeneity I 2 exceeded 90%, even after adjustment for the factors associated with antibody response. Two significant determinants were independently associated with antibody response after preventive vaccination against EVD: the Ebola vaccine platform and the Ebola strain used for antibody detection.

The use of a systematic review methodology, including solicitation of experts, allowed us to conduct exhaustive descriptive analyses on all Ebola vaccinated groups in NHP or humans published in the literature up to January 2017. Our descriptive results showed an extreme variability of study designs and features, especially in nonhuman primate trials. This variability is related to the recentness of the research topic. The higher variability within nonhuman primate studies compared to human trials is easily explained by the process of vaccine development, which selects for further clinical trials only the subset of candidate vaccines proven to be immunogenic in nonhuman primates. The comparison of antibody response levels between humans and nonhuman primate only had an indicative purpose. It is indeed difficult to compare these very different models, mostly because of potential multiple confounding factors.

Due to the low sample size of each group of nonhuman primates, we decided to restrict heterogeneity analyses to human groups. Human groups with small sample size were excluded, since their between-group variance would have been too low to contribute to the meta-regression model. It was not possible to pool small groups together because of high heterogeneity in the factors likely to influence the antibody response (vaccine and population characteristics, and measure of antibody response). The threshold of at least 8 individuals per group allowed us to include the majority of human groups in the meta-regression. Sensitivity analyses using a threshold of 10 individuals led to the same final results.

The very high heterogeneity between vaccination groups could be explained by various reasons. Firstly, some factors influencing the antibody response may be missing, for instance, genetic factors that are influencing the immunogenicity of the vaccines [START_REF] Sridhar | Clinical development of Ebola vaccines[END_REF]. Secondly, the analysis of grouped data, due to unavailability of individual data for the groups included in our meta-regression model, led to a lack of precision in the estimation of influence of factors on antibody response, and also in the evaluation of antibody response heterogeneity across vaccination groups. Thirdly, the enzyme-linked immunosorbent assay (ELISA) measuring relative antibody concentration of immunoglobulin G against EBOV glycoprotein used in the different trials could have a variation of its precision [START_REF] Logue | Use of the Filovirus Animal Non-Clinical Group (FANG) Ebola virus Immuno-assay requires fewer study participants to power a study than the Alpha Diagnostic International assay[END_REF]. Lastly, the extreme variability of study designs certainly explains parts of the high between-group variance for antibody response observed in our results.

Despite the major between-group heterogeneity in our metaregression model, two factors significantly associated with antibody response variability could be identified. The Ebola strain used for antibody detection seems to influence the results of ELISA tests. This demonstrates the importance of harmonisation for the measurement methods used in vaccines evaluations, and highlights the difficulty in directly comparing published results across several trials. The Ebola vaccine platform was also strongly associated with antibody response.

For the other factors studied in our meta-analysis, no association was found with the antibody response variability. In particular, the vaccine dosage did not have any significant influence on the level of the antibody response in our results. We acknowledge that the use of a binary variable may have limited the ability to detect a dose-effect in the meta-regression. However, the regression result is consistent with the descriptive results that also did not suggest a clear dose-immunogenicity relationship within a given vaccine platform.

No population characteristic was independently associated with the antibody response after Ebola vaccination. It may be possible that the low diversity of the population, which is directly related to the strict criteria for selection of trial participants, prevented the identification of a potential impact of these population characteristics on the antibody response.

Conclusion

Our findings show that there are still significant uncertainties in the determinants of the antibody response after preventive vaccination against Ebola virus disease. This emphasises the interest of harmonizing measurement methods and study designs. Furthermore, it indicates the impossibility to directly compare results from one published study to another or to extrapolate results, due to considerable variations in studies features. Assessment of immunogenicity between Ebola vaccines needs randomised controlled multi-arm trials, as performed in PREVAIL study (NCT02344407) and PREVAC study (NCT02876328).

Figure 1 .

 1 Figure 1. Flow chart for study/vaccination group selection.

Figure 2 .

 2 Figure 2. Description of the number of vaccine clinical trials against Ebola per country. The Ring trial, single phase 3 trial (Guinea), has been excluded from the systematic review. Several other vaccine clinical trials against Ebola are currently ongoing worldwide but only published trials are reported in the figure.

Figure 3 .

 3 Figure 3. Number of vaccination groups of humans and of nonhuman primates, according to the number of individuals by group.

Figure 4 .

 4 Figure 4. Forest plot of antibody titre after Ebola vaccination for each vaccination group by vaccine platform. Colour codes indicate dose levels within a given platform. GP: glycoprotein. PFU: plaque forming unit. VP: viral particle. TCID: tissue culture infectious dose. References for Figure 4: 1: Agnandji 2016, VSV vaccine (3.10 6 PFU) with Zaire insert, Germany, detection with Zaire Kikwit GP 2: Agnandji 2016, VSV vaccine (2.10 7 PFU) with Zaire insert, Germany, detection with Zaire Kikwit GP 3: Agnandji 2016, VSV vaccine (3.10 5 PFU) with Zaire insert, Gabon, detection with Zaire Kikwit GP 4: Agnandji 2016, VSV vaccine (3.10 6 PFU) with Zaire insert, Gabon, detection with Zaire Kikwit GP 5: Agnandji 2016, VSV vaccine (3.10 6 PFU) with Zaire insert, Kenya, detection with Zaire Kikwit GP 6: Agnandji 2016, VSV vaccine (2.10 7 PFU) with Zaire insert, Kenya, detection with Zaire Kikwit GP 7: Agnandji 2016, VSV vaccine (1.10 7 PFU) with Zaire insert, Switzerland, detection with Zaire Kikwit GP 8: Agnandji 2016, VSV vaccine (5.10 7 PFU) with Zaire insert, Switzerland, detection with Zaire Kikwit GP 9: De Santis 2016, ChAd3 vaccine (2.5.10 10 VP) with Zaire Mayinga insert, Switzerland, detection with Zaire Mayinga GP 10: De Santis 2016, ChAd3 vaccine (5.10 10 VP) with Zaire Mayinga insert, Switzerland, detection with Zaire Mayinga GP

Table 1

 1 Main characteristics of the preclinical studies and clinical trials included in the systematic review.

	Title	First author Year of	Population and study	Vaccine(s)	Measurement of
			publication	features		antibody response
	Phase 1 Trials of rVSV Ebola Vaccine in Africa	Agnandji	2016	Humans (Germany,	Recombinant VSV-GP(Zaire), single injection,	Antibodies anti GP
	and Europe (Agnandji et al., 2016)			Switzerland, Gabon,	IM, 300 000 to 50 million PFU	(Kikwit), D28 or
				Kenya), phase 1,		D180
				randomization and
				placebo
	Successful topical respiratory tract	Bukreyev	2007	NHP: rhesus monkeys;	Recombinant HPIV3(+/À modified)-GP +/À NP	Antibodies anti
	immunization of primates against Ebola			placebo	(Zaire Mayinga), single injection +/À boost	virion, D28 (or D39
	virus (Bukreyev et al., 2007)				D28, IN + IT, 4 to 20 million TCID50	after boost)
	Mucosal parainfluenza virus-vectored vaccine	Bukreyev	2010	NHP: rhesus monkeys	Recombinant HPIV3-GP(Zaire Mayinga),	Antibodies anti
	against Ebola virus replicates in the			(+/À HPIV3	boost D28, IN + IT, 20 million PFU	virion, D28
	respiratory tract of vector-immune			seropositive); placebo
	monkeys and is immunogenic (Bukreyev
	et al., 2010)
	Safety and immunogenicity of a chimpanzee	De Santis	2016	Humans (Switzerland),	Recombinant ChAd3-GP(Zaire Mayinga),	Antibodies anti GP
	adenovirus-vectored Ebola vaccine in			phase 1/2,	single injection, IM, 25 to 50 billion VP	(Mayinga), D28,
	healthy adults: a randomised, double-blind,			randomization and		results in EC90
	placebo-controlled, dose-finding, phase 1/			placebo
	2a study (De Santis et al., 2016)
	Respiratory tract immunization of non-	DiNapoli	2010	NHP: rhesus monkeys;	Recombinant NDV-GP(Zaire Mayinga) or	Antibodies anti
	human primates with a Newcastle disease			no placebo	HPIV3-GP(Zaire Mayinga), boost D28, IN + IT,	virion (Mayinga),
	virus-vectored vaccine candidate against				20 million PFU	D28
	Ebola virus elicits a neutralizing antibody
	response (DiNapoli et al., 2010)
	A Monovalent Chimpanzee Adenovirus Ebola	Ewer	2016	Humans (United	Recombinant ChAd3-GP(Zaire Mayinga) 10 to	Antibodies anti GP
	Vaccine Boosted with MVA (Ewer et al.,			Kingdom), phase 1, no	50 billion VP, boost between D7 and D46 with	(Mayinga) or anti
	2016)			randomization, no	recombinant MVA-GP(Zaire Mayinga + Sudan	virion (Makona)
				placebo	Gulu)/NP(Taï Forest) 150 to 300 millions PFU,
					IM
	Vesicular stomatitis virus-based vaccines	Geisbert	2008	NHP: cynomolgus	Recombinant VSV-GP(Zaire Kikwit), single	Antibodies anti
	protect nonhuman primates against aerosol			macaques; placebo	injection, 20 million PFU	virion (Kikwit), D14
	challenge with Ebola and Marburg viruses					or D27
	(Geisbert et al., 2008a,b)
	Vesicular stomatitis virus-based ebola vaccine	Geisbert	2008	NHP: rhesus monkeys	Recombinant VSV-GP(Zaire Mayinga), single	Antibodies anti
	is well-tolerated and protects			(SHIV infected);	injection, IM, 10 million PFU	virion (Mayinga),
	immunocompromised nonhuman primates			placebo		D14
	(Geisbert et al., 2008a,b)
	Single-injection vaccine protects nonhuman	Geisbert	2009	NHP: cynomolgus	Recombinant VSV-GP(Zaire Mayinga and/or	Antibodies anti
	primates against infection with marburg			macaques and rhesus	Sudan Boniface +/À Marburg), single injection	virion, between
	virus and three species of ebola virus			monkeys; placebo	+/À boost D14, IM, 10 to 20 million PFU	D14 and D28
	(Geisbert et al., 2009)
	Recombinant adenovirus serotype 26 (Ad26)	Geisbert	2011	NHP: cynomolgus	Recombinant Ad5, Ad26, or Ad35, or prime	Antibodies anti GP,
	and Ad35 vaccine vectors bypass immunity			macaques (+/À Ad5	Ad26 + boost Ad35 D28 -GP (Zaire + Sudan	D21, results in EC90
	to Ad5 and protect nonhuman primates			seropositive); placebo	Gulu), IM, 20 to 200 billion VP
	against ebolavirus challenge (Geisbert et al.,
	2011)
	Codon-optimized filovirus DNA vaccines	Grant-Klein 2015	NHP: cynomolgus	Vaccin ADN-GP(Zaire +/À Sudan, Reston et	Antibodies anti GP
	delivered by intramuscular electroporation			macaques; placebo	Marburg), 3 injections (28 jours apart),	(Mayinga) DTM or
	protect cynomolgus macaques from lethal				electroporation IM, 500 mg to 2 mg	DMuc, D28
	Ebola and Marburg virus challenges (Grant-
	Klein et al., 2015)
	Demonstration of cross-protective vaccine	Hensley	2010	NHP: cynomolgus	Vaccin ADN-GP(Zaire Mayinga + Sudan Gulu),	Antibodies anti GP,
	immunity against an emerging pathogenic			macaques; placebo	4 injections IM, 4 mg (28 to 42 days apart +/À	D21 or D371,
	Ebolavirus Species (Hensley et al., 2010)				boost D371 recombinant Ad5-GP (Zaire	results in EC90
					Mayinga) IM 100 billion VP
	Venezuelan equine encephalitis virus replicon	Herbert	2013	NHP: cynomolgus	VRP GP(Zaire Kikwit +/À Sudan Boniface),	Antibodies anti GP,
	particle vaccine protects nonhuman			macaques; placebo	single injection, IM, 10 to 20 billion FFU	D28
	primates from intramuscular and aerosol
	challenge with ebolavirus (Herbert et al.,
	2013)
	The effect of dose on the safety and	Huttner	2015	Humans (Switzerland),	Recombinant VSV-GP(Zaire), single injection,	Antibodies anti GP,
	immunogenicity of the VSV Ebola candidate			phase 1/2,	IM, 300 000 PFU	D28
	vaccine: a randomised double-blind,			randomization and
	placebo-controlled phase 1/2 trial (Huttner			placebo
	et al., 2015)
	Live attenuated recombinant vaccine protects	Jones	2005	NHP: cynomolgus	Recombinant VSV-GP(Zaire Mayinga), single	Antibodies anti
	nonhuman primates against Ebola and			macaques; placebo	injection, IM, 10 million PFU	virion, D28
	Marburg viruses (Jones et al., 2005)
	Phase 2 Placebo-Controlled Trial of Two	Kennedy	2017	Humans (Liberia),	Recombinant ChAd3-GP(Saire) 100 billion VP	Antibodies anti GP
	Vaccines to Prevent Ebola in Liberia			phase 2,	or VSV-GP(Zaire Kikwit) 20 million PFU, single	(Kikwit), D28
	(Kennedy et al., 2017)			randomization and	injection, IM
				placebo
	Safety and immunogenicity of Ebola virus and	Kibuuka	2015	Humans (Uganda),	Vaccin ADN GP(Zaire + Sudan +/À Marburg), 3	Antibodies anti GP,
	Marburg virus glycoprotein DNA vaccines			phase 1b;	injections, IM, 4 mg	D28
	assessed separately and concomitantly in			randomization and
	healthy Ugandan adults: a phase 1b,			placebo

Table 1 (

 1 Continued)

	Title	First author Year of	Population and study	Vaccine(s)	Measurement of
			publication	features		antibody response
	Safety and immunogenicity of DNA vaccines	Sarwar	2015	Humans (USA), phase	Vaccin ADN GP(Zaire + Sudan), 3 injections	Antibodies anti GP,
	encoding Ebolavirus and Marburgvirus			1, no randomization	(28 days apart) + boost D168, IM, 4 mg	D28
	wild-type glycoproteins in a phase I clinical			and no placebo
	trial (Sarwar et al., 2015)
	Chimpanzee adenovirus vaccine generates	Stanley	2014	NHP: cynomolgus	Recombinant ChAd3-GP(Zaire + Sudan), 1 to	Antibodies anti GP,
	acute and durable protective immunity			macaques; placebo	10 billion VP or recombinant ChAd3-GP	D21, results in EC90
	against ebolavirus challenge (Stanley et al.,				(Zaire + Sudan) or recombinant MVA-GP
	2014)				(Zaire + Sudan) 100 million VP, single
					injection, IM
	Development of a preventive vaccine for Ebola	Sullivan	2000	NHP: cynomolgus	DNA vaccine GP/NP(Zaire) + GP(Sudan + Taï	Nature of viral
	virus infection in primates (Sullivan et al.,			macaques; placebo	Forest), 3 injections, 4 mg (28 days apart),	antigen non
	2000a,b)				boost D84 recombinant Ad5-GP(Z) 10 billion	specified, D28
					PFU, IM
	Accelerated vaccination for Ebola virus	Sullivan	2003	NHP: cynomolgus	Recombinant Ad5-GP/NP(Zaire) +/À boost	Antibodies anti
	haemorrhagic fever in non-human primates			macaques; placebo	D63, IM, 2000 billion VP	virion, between D7
	(Sullivan et al., 2003)					and D63
	CD8+ cellular immunity mediates rAd5	Sullivan	2011	NHP: cynomolgus	Recombinant Ad5-GP(Zaire), single injection,	Antibodies anti GP,
	vaccine protection against Ebola virus			macaques; placebo	IM, 10 billion VP	day of
	infection of nonhuman primates (Sullivan					measurement non
	et al., 2011)					specified, results in
						EC90
	Vaccine to confer to nonhuman primates	Swenson	2008	NHP: cynomolgus	Recombinant Ad5-GP/NP(Zaire) + GP(Sudan	Antibodies anti
	complete protection against multistrain			macaques; placebo	Boniface), boost D63, IM, 40 billion PFU	virion, D14 after
	Ebola and Marburg virus infections					prime and D21
	(Swenson et al., 2008)					after boost
	Use of ChAd3-EBO-Z Ebola virus vaccine in	Tapia	2016	Humans (Mali), phase	Recombinant ChAd3-GP(Zaire), 10 to 100	Antibodies anti GP,
	Malian and US adults, and boosting of			1, randomization and	billion VP, boost D97 recombinant MVA-GP	D28
	Malian adults with MVA-BN-Filo: a phase 1,			placebo	(Zaire + Sudan + Marburg) + NP (Taï Forest)
	single-blind, randomised trial, a phase 1b,				200 millions PFU, IM
	open-label and double-blind, dose-
	escalation trial, and a nested, randomised,
	double-blind, placebo-controlled trial
	(Tapia et al., 2016)
	Ebola virus-like particle-based vaccine	Warfield	2007	NHP: cynomolgus	VLP GP/VP40/NP(Zaire), 2 injections (42 days	Antibodies anti
	protects nonhuman primates against lethal			macaques; placebo	apart), boost D42, IM, 250 mg	virion, D42
	Ebola virus challenge (Warfield et al., 2007)
	Vaccinating captive chimpanzees to save wild	Warfield	2014	NHP: chimpanzee; no	VLP (with adjuvant: IDC-1001 ou CpG) GP/	Antibodies anti
	chimpanzees (Warfield et al., 2014)			placebo	VP40/NP(Zaire), 2 injections (29 days apart),	GPDTM or VP40,
					boost D27, IM, 3 mg	between D27 and
						D29, results in
						EC50
	Homologous and heterologous protection of	Warfield	2015	NHP: cynomolgus	VLP GP/VP40/NP(Zaire et/ou Sudan), boost	Antibodies anti
	nonhuman primates by Ebola and Sudan			macaques; placebo	D42, IM, 3 mg	GPDTM or VP40,
	virus-like particles (Warfield et al., 2015)					between D14 and
						D28
	Immune parameters correlate with protection	Wong	2012	NHP: cynomolgus	Recombinant VSV-GP(Zaire Mayinga), single	Antibodies anti GP,
	against ebola virus infection in rodents and			macaques; placebo	injection, IM or IT or PO, 20 millions PFU	D28
	nonhuman primates (Wong et al., 2012)
	An Adenovirus Vaccine Expressing Ebola	Wu	2016	NHP: cynomolgus	Recombinant Ad5-GP(Zaire Makona), single	Antibodies anti GP,
	Virus Variant Makona Glycoprotein Is			macaques; placebo	injection, IM, 40 or 200 billion VP	D28
	Efficacious in Guinea Pigs and Nonhuman
	Primates (Wu et al., 2016)
	Safety and immunogenicity of a novel	Zhu	2015	Humans (China),	Recombinant Ad5-GP(Zaire Makona), single	Antibodies anti GP
	recombinant adenovirus type-5 vector-			phase 1,	injection, IM, 40 to 160 billions VP	(Makona), D28
	based Ebola vaccine in healthy adults in			randomization and
	China: preliminary report of a randomised,			placebo
	double-blind, placebo-controlled, phase 1
	trial (Zhu et al., 2015)
	Safety and immunogenicity of a recombinant	Zhu	2017	Humans (Sierra	Recombinant Ad5-GP(Zaire Makona), single	Antibodies anti GP,
	adenovirus type-5 vector-based Ebola			Leone), phase 2;	injection, IM, 40 or 160 billion VP	D28
	vaccine in healthy adults in Sierra Leone: a			randomization and
	single-centre, randomised, double-blind,			placebo
	placebo-controlled, phase 2 trial (Zhu et al.,
	2017)

Table 2

 2 Main characteristics of included non-human primates (NHP) groups.

	Characteristic	Vaccination schedule			All NHP groups
		No boost (n = 98)		Boost (n = 30)		n = 128
	Vaccine platform
	DNA vaccine (plasmid)	6	6.1%	0	0.0%	6	4.7%
	Adenovirus 26	4	4.1%	0	0.0%	4	3.1%
	Adenovirus 26 then adenovirus 35	0	0.0%	1	3.3%	1	0.8%
	Adenovirus 35	4	4.1%	0	0.0%	4	3.1%
	Adenovirus 5	8	8.2%	3	10.0%	11	8.6%
	DNA vaccine (plasmid)/adenovirus 5	0	0.0%	2	6.7%	2	1.6%
	CAdVax	6	6.1%	2	6.7%	8	6.2%
	Chimpanzee adenovirus 3	2	2.0%	0	0.0%	2	1.6%
	Chimpanzee adenovirus 63	1	1.0%	0	0.0%	1	0.8%
	HPIV3	12	12.2%	7	23.3%	19	14.8%
	MVA	1	1.0%	0	0.0%	1	0.8%
	NDV	1	1.0%	1	3.3%	2	1.6%
	RhCMV	1	1.0%	0	0.0%	1	0.8%
	Whole-virus vaccine	4	4.1%	0	0.0%	4	3.1%
	VLP	21	21.4%	10	33.3%	31	24.2%
	VLP Kunjin	1	1.0%	1	3.3%	2	1.6%
	VRP VEEV	3	3.1%	1	3.3%	4	3.1%
	VSV	23	23.5%	2	6.7%	25	19.5%
	Route of administration
	Intramuscular	78	79.6%	21	70.0%	99	77.3%
	Other routes	20	20.4%	9	30.0%	29	22.7%
	Vaccine insert: Ebola species
	Monovalent Zaire	63	64.3%	17	56.7%	80	62.5%
	Monovalent no Zaire	6	6.1%	2	6.7%	8	6.2%
	Monovalent no Zaire + monovalent Zaire	0	0.0%	1	3.3%	1	0.8%
	Monovalent no Zaire + multivalent	0	0.0%	1	3.3%	1	0.8%
	Multivalent	29	29.6%	7	23.3%	36	28.1%
	Multivalent + monovalent Zaire	0	0.0%	2	6.7%	2	1.6%
	Vaccine insert: Ebola strain (only for Zaire species)
	Mayinga	34	64.2%	13	76.5%	47	67.1%
	Kikwit	16	30.2%	4	23.5%	20	28.6%
	Makona	3	5.7%	0	0.0%	3	4.3%
	Missing data	45	-	13	-	58	-
	Nonhuman primates species
	Cynomolgus macaques	65	66.3%	17	56.7%	82	64.1%
	Chimpanzees	10	10.2%	2	6.7%	12	9.4%
	Rhesus macaques	22	22.4%	10	33.3%	32	25.0%
	African green monkeys	1	1.0%	1	3.3%	2	1.6%
	Year of publication
	Publication < 2014	49	50.0%	14	46.7%	63	49.2%
	Publication ! 2014	49	50.0%	16	53.3%	65	50.8%
	Time interval between last injection and antibody measure
	Mean [standard deviation]	29.1	[363]	25.5	[762]	28.3	[319]
	Missing data	3	-	0	-	3	-
	Antibody measurement method
	Maximal dilution	65	66.3%	18	60.0%	83	64.8%
	Effective concentration 90 (EC90)	15	15.3%	2	6.7%	17	13.3%
	Effective concentration 50 (EC50)	18	18.4%	10	33.3%	28	21.9%
	Antigen used for antibody detection: nature
	Glycoprotein (GP)	55	56.7%	7	24.1%	62	49.2%
	Other nature (virion, viral protein 40)	42	43.3%	22	75.9%	64	50.8%
	Missing data	1	-	1	-	2	-
	Antigen used for antibody detection: Ebola strain
	Mayinga	14	87.5%	8	100.0%	22	91.7%
	Kikwit	2	12.5%	0	0.0%	2	8.3%
	Missing data	82	-	22	-	104	-
	Similarity between strain used as vaccine insert and strain used for antibody detection
	Identical strains	12	100.0%	8	100.0%	20	100.0%
	Missing data	86	-	22	-	108	-

CAdVax: complex adenovirus-based vector, DNA: deoxyribonucleic acid, GP: glycoprotein, HPIV3: human parainfluenza virus 3, MVA: modified vaccinia Ankara, NDV: Newcastle disease virus, RhCMV: rhesus cytomegalovirus cytomegalovirus, VLP: virus-like particles, VRP VEEV: Venezuelan equine encephalitis virus replicon particle, VSV: vesicular stomatitis virus.

Table 3

 3 Main characteristics of included human groups.

	Characteristics	Vaccination schedule			All human groups
		No boost (n = 48)		Boost (n = 26)		(n = 74)
	Vaccine platform
	DNA vaccine (plasmid)	9	18.8%	1	3.8%	10	13.5%
	Adenovirus 26	3	6.2%	0	0.0%	3	4.1%
	Adenovirus 26/MVA or MVA/adenovirus 26	0	0.0%	5	19.2%	5	6.8%
	Adenovirus 5	6	12.5%	2	7.7%	8	10.8%
	Chimpanzee adenovirus 3	14	29.2%	0	0.0%	14	18.9%
	Chimpanzee adenovirus 3/MVA	0	0.0%	18	69.2%	18	24.3%
	MVA	2	4.2%	0	0.0%	2	2.7%
	VSV	14	29.2%	0	0.0%	14	18.9%
	Route of administration
	Intramuscular	48	100.0%	26	100.0%	74	100.0%
	Vaccine insert: species
	Monovalent Zaire	31	64.6%	19	73.1%	50	67.4%
	Monovalent Zaire + multivalent	0	0.0%	4	15.4%	4	5.4%
	Multivalent	17	35.4%	1	3.8%	18	24.3%
	Multivalent + monovalent Zaire	0	0.0%	2	7.7%	2	2.7%
	Vaccine insert: strain (only for Zaire species)
	Mayinga	22	71.0%	22	91.7%	44	80.0%
	Kikwit	5	16.1%	0	0.0%	5	9.1%
	Makona	4	12.9%	0	0.0%	6	10.9%
	Missing data	17	-	4	-	19	-
	Proportion of women
	Mean [standard deviation]	40%	[18%]	52%	[10%]	44%	[17%]
	Mean age (years)
	Mean [standard deviation]	34.8	[45]	34.6	[54]	34.7	[48]
	Geographic location of the study
	Africa	14	29.2%	1	3.8%	15	20.3%
	China	2	4.2%	2	7.7%	4	5.4%
	Europe	15	31.2%	22	84.6%	37	50.0%
	USA	17	35.4%	1	3.8%	18	24.3%
	Year of publication
	Publication < 2014	8	16.7%	0	0.0%	8	10.8%
	Publication ! 2014	40	83.3%	26	100.0%	66	89.2%
	Time interval between last injection and antibody measure (days)
	Mean [standard deviation]	31.2	[220]	26.1	[32]	29.3	[179]
	Antibody measurement method
	Maximal dilution	35	72.9%	24	92.3%	59	79.7%
	Effective concentration 90 (EC90)	13	27.1%	2	7.7%	15	20.3%
	Antigen used for antibody detection: nature
	Glycoprotein (GP)	45	93.8%	25	96.2%	70	94.6%
	Other nature (virion, nucleoprotein)	3	6.2%	1	3.8%	4	5.4%
	Antigen used for antibody detection: Ebola strain
	Mayinga	14	36.8%	16	72.7%	30	50.0%
	Kikwit	18	47.4%	5	22.7%	23	38.3%
	Makona	6	15.8%	1	4.5%	7	11.7%
	Missing data	10	-	4	-	14	-
	Similarity between strain used as vaccine insert and strain used for antibody detection
	Different strains	9	32.1%	6	27.3%	15	30.0%
	Identical strains	19	67.9%	16	72.7%	35	70.0%
	Missing data	20	-	4	-	24	-
	CAdVax: complex adenovirus-based vector, DNA: deoxyribonucleic acid, GP: glycoprotein, HPIV3: human parainfluenza virus 3, MVA: modified vaccinia Ankara, NDV:
	Newcastle disease virus, RhCMV: rhesus cytomegalovirus cytomegalovirus, VLP: virus-like particles, VRP VEEV: Venezuelan equine encephalitis virus replicon particle, VSV:
	vesicular stomatitis virus.

Table 4

 4 Results of a random-effect meta-regression model (with fixed intragroup variance) of determinants of antibody titre (log10) after Ebola vaccination according to characteristics of vaccine, population, and measurement techniques. Multivariate analysis. I 2 = 95.31%, R 2 = 68.45%.

	Determinants of antibody response	Estimated β [CI 95%]

Funding source

This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 115861. This Joint Undertaking receives support from the European Union's Horizon 2020 research and innovation programme and EFPIA.

The funder of the study had no role in the study design, data collection, data analysis, data interpretation, or writing the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.

Conflict of interest

This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No. 115861 This Joint Undertaking receives support from the European Union's Horizon 2020 research and innovation program and EFPIA.

Laura Richert and Rodolphe Thiebaut are also involved in the ongoing PREVAC (NCT02876328) and Ebovac2 (NCT02564523, NCT02416453) trials. Edouard Lhomme is involved in the PREVAC trial (NCT02876328).

Ethical considerations

According to the National Public Health Code, review and metaanalysis do not require ethical approval.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.ijid.2018.06.022.