Main phenolic compounds in different grape parts (expressed in g/kg of dm).
\\n\\n
More than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\\n\\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\\n\\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\\n\\nAdditionally, each book published by IntechOpen contains original content and research findings.
\\n\\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\\n\\n\\n\\n
\\n"}]',published:!0,mainMedia:null},components:[{type:"htmlEditorComponent",content:'
Simba Information has released its Open Access Book Publishing 2020 - 2024 report and has again identified IntechOpen as the world’s largest Open Access book publisher by title count.
\n\nSimba Information is a leading provider for market intelligence and forecasts in the media and publishing industry. The report, published every year, provides an overview and financial outlook for the global professional e-book publishing market.
\n\nIntechOpen, De Gruyter, and Frontiers are the largest OA book publishers by title count, with IntechOpen coming in at first place with 5,101 OA books published, a good 1,782 titles ahead of the nearest competitor.
\n\nSince the first Open Access Book Publishing report published in 2016, IntechOpen has held the top stop each year.
\n\n\n\nMore than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\n\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\n\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\n\nAdditionally, each book published by IntechOpen contains original content and research findings.
\n\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\n\n\n\n
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Grape and wine phenols represent a large family of compounds with a great diversity of chemical structures and degrees of complexity. The term “polyphenols” or “phenolics” is used to define a group of plant secondary metabolites that presents one or more than one hydroxyl (▬OH) groups attached to one or more benzene rings [1].
Polyphenols are synthetized by phenylpropanoid pathway, being the amino acid phenylalanine (a shikimate pathway product) their common precursor. They can be divided into flavonoid (anthocyanins, flavan-3-ols, flavonols, flavanones, flavanonols, flavones, and chalcones) and non-flavonoid (hydroxybenzoic and hydroxycinnamic acids and stilbenes) families [2].
These compounds are critically important for wine quality, due to their contribution to their sensory properties: color, taste, mouthfeel, flavor, astringency, and bitterness [3, 4]. For this reason, the understanding of the relationship between wine quality and its phenolic composition is considered, nowadays, one of the major challenges in enology research.
Furthermore, fermentation, maturation, and/or aging of wine may be performed in contact with oak wood. Spontaneous clarification, slow and continuous oxygen diffusion through the oak wood pores (for barrels and casks), and extraction of many volatile and nonvolatile (mainly ellagitannins) compounds are observed. As a result, wine undergoes a modulation of its quality and complexity with regard to aroma, structure, astringency, bitterness, persistence, and color stability [5].
The objective of this book chapter is to examine the key phenolic compounds in grapes and in oak wood used for the maturation of wine. Likewise, the evolution of these compounds during winemaking and wine aging and their impact in the sensory properties of wine will be discussed.
The most important grape polyphenols comprise anthocyanins, flavan-3-ols, proanthocyanidins and flavonols (flavonoid family), and phenolic acids and stilbenes (non-flavonoid family). Each family can be present in their free or conjugated forms, differing by their hydroxylation level and by the substitution of the hydroxy groups (methylation, glycosylation, acylation) and even forming adducts between them (e.g., phenolic acids with anthocyanins; condensed tannins). This fact explains the great chemical diversity of polyphenols in grapes [6].
It is noteworthy that polyphenolic composition in grapes is highly affected by different factors such as viticulture practices, environmental conditions (soil, climate), and pathogen attacks [7]. Although, one of the most important factors is undoubtedly the varietal or genetic differences [8] as well as the winemaking process.
Flavonoids are basically formed by a structure of 15 carbons (C6-C3-C6) divided in 2 aromatic rings, A and B, which are joined by a 3-carbon chain that is part of a heterocyclic C ring (Figure 1). Depending on the oxidation state of C ring, this family can be subdivided in anthocyanins, flavan-3-ols, or flavonols.
General chemical structure of flavonoid family.
Structurally, anthocyanins are mainly present in nature in the form of heterosides. The aglycone form of anthocyanins, also called anthocyanidin, is based on the flavylium or 2-phenylbenzopyrilium ion having hydroxyl and methoxyl groups in different positions.
Anthocyanins are the most important natural pigments in wine grapes. These compounds are predominately accumulated in skins of red grapes during the ripening, and they are also present in the flesh of “teinturier” varieties [9]. In addition, it has been recently demonstrated that certain white grape cultivars (Sauvignon Blanc, Riesling, and Chardonnay) can contain measurable traces of anthocyanins [10]. Several factors can influence the anthocyanin biosynthesis in grapes such as origin and type of the grape vine, degree of maturity, and weather conditions like temperature, water availability, or the light exposure and intensity [11].
Regarding total anthocyanins, their quantities vary between 11.47 and 29.83 g/kg of dry matter (dm) in red grape skins [12] (Table 1). The principal individual anthocyanins in Vitis vinifera cultivars are the 3-O-monoglucosides (glucose linked through glucosidic bonds at the C3 positions of C ring) of delphinidin, cyanidin, petunidin, peonidin, and malvidin (Figure 2). Among these, malvidin-3-O-glucoside is generally the most abundant with values of 4.12–10.19 g/kg dm [14]. More recently, He and co-workers demonstrated for the first time the presence of pelargonidin-3-O-glucoside at trace levels on berry skins of Cabernet Sauvignon and Pinot noir cultivars [20]. Moreover, the monoglucoside forms can be acylated at the C6″ position of the glucose moiety with both aromatic (p-coumaric, caffeic, ferulic, and sinapic acid) or aliphatic acids (acetic, malic, malonic, oxalic, and succinic acid). The most common acylated anthocyanins in V. vinifera grape includes 3-O-(6″-p-coumaroyl)-glucosides, 3-O-(6″-acetyl)-glucosides, and 3-O-(6-caffeoyl)-glucosides of delphinidin, petunidin, peonidin, and malvidin [8, 12, 14, 21]. To go further, even anthocyanin dimers (malvidin-3-O-glucoside dimer and malvidin-3-O-glucoside-peonidin-3-O-glucoside) have been identified in grape skins [8, 22]. The presence of these acetylated forms is important for the color stabilization and intensity of wines [23]. The color intensity increases with the number of substituted groups on the B ring (di-oxygenated forms are redder, while tri-oxygenated are more purple) and with the replacement of hydroxyl by methoxyl groups (i.e., malvidin has the darkest color). Moreover, methoxylated anthocyanins (malvidin and peonidin) are more stable than hydroxylated ones to environmental and viticultural factors [24]. Additionally, anthocyanins can be found as 3,5-O-diglucosides or acylated 3,5-O-diglucosides, which are considered as marker compounds of non-V. vinifera species or hybrid red grapes [25].
Compound | g/kg dm | Grape variety | References | ||
---|---|---|---|---|---|
Pomace | Seeds | Skins | |||
Total polyphenol contenta | 19–40.5 | 36.6–88.7 | 20.2–52.3 | Grenache, Syrah, Carignan noir, Mourvèdre, Counoise | [13] |
Anthocyanins | |||||
Total | 11.47–29.82 | 11–47–29.82 | ND | Cabernet Mitos, Lemberger, Spätburgunder, Schwarzriesling, Trollinger | [12, 14] |
Delphidin-3-O-glucoside | 0.44–1.11 | 0.44–1.11 | ND | ||
Cyanidin-3-O-glucoside | 1.51–3.81 | 1.51–3.81 | ND | ||
Petunidin-3-O-glucoside | 0.53–1.34 | 0.53–1.34 | ND | ||
Peonidin-3-O-glucoside | 0.99–2.49 | 0.99–2.49 | ND | ||
Malvidin-3-O-glucoside | 4.12–10.19 | 4.12–10.19 | ND | ||
Total acetylated | 3.88–10.88 | 3.88–10.88 | ND | ||
Flavan-3-ols/proanthocyanidins | |||||
Catechin | 0–0.3 | 2.14–2.42 | 0–0.3 | Grenache, Syrah, Carignan noir, Mourvèdre, Counoise Cabernet Sauvignon, Chardonnay, Sauvignon blanc, Moscatel de Grano Menudo, Gewürztraminer, Riesling, Viognier, Merlot, Cencibel | [13, 15, 16, 17, 18, 19] |
Epicatechin | 0–0.2 | 0.88–1.60 | 0–0.13 | ||
Epigallocatechin | 0–0.05 | 0.05 | ND | ||
Epigallocatechin-3-O-gallate | 0–0.007 | 0.06–0.07 | ND | ||
Epicatechin-3-O-gallate | 0.003 | 0.25–0.31 | 0.04 | ||
Procyanidin B1 | 0.11–0.60 | 0.14–0.17 | 0.18–0.6 | ||
Procyanidin B2 | 0.01–0.84 | 0.04–0.18 | 0.01–0.84 | ||
Total tannins | 39.1–105.8 | 62.3–167.8 | 44.9–73.0 | ||
Flavonols | |||||
Total | 0.03–0.63 | 0.48–0.63 | 0.02–0.05 | Merlot Weisser Riesling | [14] |
Phenolic acids | |||||
Total | 0.03–8.31 | 0.10–0.11 | 0.17–8.23 | Cabernet Sauvignon, Merlot, Cabernet Mitos | [12, 14, 19] |
Gallic acid | 0.03–0.11 | 0.03 | 0.01–0.11 | ||
Coutaric acid | 0–1.23 | 0.03–1.23 | — | ||
Caftaric acid | 0–6.97 | 0.11–6.97 | — |
Main phenolic compounds in different grape parts (expressed in g/kg of dm).
Gallic acid equivalents (GAE) per g dry weight.
Chemical structures of major grape phenolic compounds.
In general, anthocyanin concentration is maximized under nonirrigated conditions in all cultivars, but anthocyanin profile and relative distribution of individual anthocyanins among irrigation treatments are influenced principally by the cultivar. In fact, Cabernet Sauvignon, Merlot, Syrah, and Tempranillo are characterized by a major proportion of malvidin forms, while in Nebbiolo (Italian cultivar) peonidin-3-O-glucoside is the most prevalent anthocyanin [11]. Other varieties, for example, Pinot noir, red Chardonnay, and pink Sultana (white red-colored mutants), are not able to synthetize acetylated anthocyanins [26]. In consequence, the anthocyanins profile in grapes can be used as an authentication tool of varietal wines [27].
Flavan-3-ols are monomeric flavonoids formed by a benzopyran unit (rings A and C) with an aromatic cycle (ring B) linked to the carbon C-2 of the pyranic cycle (ring C). The presence of two chiral centers on the molecule (C2 and C3) gives rise to four possible configurations for a single monomer. These monomeric structures may be joined together forming dimers, oligomers (3–1o units of flavan-3-ols), and polymers (more than 1o units of flavan-3-ols). All these more complex structures are so-called condensed tannins. If they are formed by (+)-catechin and (−)-epicatechin and their gallic esters, they are named procyanidins, while when they are constituted by (+)-gallocatechin and (−)-epigallocatechin and their galloylated derivatives, the term used is prodelphinidins [28].
They are located in all grape clusters solid parts (skins, seeds, stalks) and are responsible for the stabilization of wines’ both color and sensory characteristics due to their astringent and bitter properties [29]. Five monomeric flavan-3-ols are commonly present in grapes (Figure 2): (+)-catechin and its stereoisomer (−)-epicatechin as the predominant ones in seeds (2.14–2.44 g/kg dm and 0.88–1.60 g/kg dm, respectively) (Table 1) (+)-gallocatechin, (−)-epigallocatechin, and (+)-catechin-3-O-gallate [13, 15, 16, 17, 18, 19].
As explained above, condensed tannins are oligomers of flavan-3-ol monomer units. These units can be linked by C-4 → C-6 or C-4 → C-8 bonds, so-called B-type proanthocyanidins. A-type condensed tannins are characterized by the presence of a second interflavonoid bond by C▬O oxidative coupling (C-2 → O-7 on the basic flavan-3-ol unit) [28]. B-type proanthocyanidins, and in particular, dimers as B1, B2, B3, and B4 or trimer C1 are mainly located in grape skins (0.01–0.86 g/kg dm) and, in a lower extent, in seeds (0.04–0.18 g/kg dm) [16, 17, 18]. On the contrary, complex procyanidins (n > 3) are more abundant in seeds (58–163 g/kg dm) than in skins (45–71 g/kg dm) (Table 1).
Tannins’ structure is characterized by the nature of its constitutive extension and terminal units, its mean degree of polymerization (mDP; average number of units in the polymer), and its degree of galloylation (%G; percentage of subunits bearing gallic acid esters). In the case of skins, the percentage of (−)-epigallocatechin (EGC) or also called the percentage of prodelphinidins (%P) is also used for characterization purposes. Condensed tannins with different mDPs may have different organoleptic properties. Generally, astringency increases with tannin concentration, molecular size, and %G [29]. Polymerized procyanidins are increasingly reactive with proteins and, therefore, have a more important astringent character [30]. Proanthocyanidins’ molecular size could also affect bitterness since monomers are considered to be more bitter than oligomers and polymers. Therefore, the estimation of both mDP and %G of procyanidins could be a useful parameter to evaluate the type of procyanidins present in a sample.
The quantity of flavan-3-ols and proanthocyanidins varies during ripening being higher at flowering and lower as the grapes maturate [31]. Both flavan-3-ols and proanthocyanidins are the major polyphenolic compounds in V. vinifera grapes. The greatest content is observed in seeds (62–168 g/kg dm) followed by skins (45–73 g/kg dm) (Table 1). This amount can be also variable depending on the grape variety and vintage [13].
Very recently, a new condensed tannin called “crown” proanthocyanidin tetramer has been isolated for the first time in grape skins of Cabernet Sauvignon cultivar. This tetramer is totally absent in seeds differentiating it from the rest of proanthocyanidins. This name “crown” is associated to an unusual macrocyclic carbon skeleton that has never been characterized before in the plant kingdom [32].
Flavonols constitute a group of flavonoids, which have the peculiarity to present a double bound between C2 and C3 and a hydroxyl group in C3. They vary in color from white to yellow and possess an important role in the color stabilization of young red wines, through copigmentation interaction with anthocyanins [3], and in the sensory perception of astringency and bitterness [33].
Conventionally, flavonols are present in berry skins of both white and colored grapes, and their total flavonoid content varies notably depending on cultivars and ripening stage [34]. In relation with cultivars, more quantities of flavonols have been reported, for example, in V. vinifera French varieties (Syrah, Petit Verdot, Cabernet Sauvignon, and Merlot), than with Spanish ones (Tempranillo, Garnacha, and Garnacha Tintorera) [35]. The total amount of flavonols in grapes varies from 1 to 80 mg/kg of fresh berry, being the red cultivars regularly richer than the white ones [35, 36].
Flavonols are found in grape berry skins in 3-O-glycoside forms. The main flavonols reported in red grapes are the dihydroxylated quercetin-3-O-glucoside and 3-O-glucuronide and the trihydroxylated myricetin 3-O-glucoside. In addition, other compounds such as kaempferol and the methylated isorhamnetin, laricitrin, and syringetin 3-O-glucosides have also been identified [35]. Furthermore, kaempferol and laricitin-3-O-galactosides, kampferol-3-O-glucuronide, and even quercetin and siringetin-3-O-(6″-acetyl)-glucoside have been identified in Cabernet Sauvignon grapes in lower quantities [37]. Interestingly and more recently, laricitrin-3-O-galactoside and syringetin-3-O-galactoside were reported in red grapes for the first time. With regard to white grapes cultivars, myricetin, laricitrin, and syringetin have not been identified [36].
Phenolic acids can be classified in two main groups: hydroxybenzoic acids (C6-C1) and hydroxycinnamic acids (C6-C3). This family is found in skins, pulp, and seeds of grapes, being generally most numerous in skins (0.2–8.2 g/kg dm) (Table 1). The quantities of total hydroxycinnamic or hydroxybenzoic acids in grape skins vary depending on cultivar and origin. For example, hydroxycinnamic acids are more predominant in V. vinifera East Asian or North American grapes than in European grapes in which these phenolic acids are only present in trace levels. However, the hydroxybenzoic acid amounts are similar between cultivars [38].
Individually, the most important hydroxybenzoic acids in grapes are gallic, vanillic, and syringic acids. Predominantly present in grape seeds, gallic acid is considered the most important phenolic acid (100–230 mg/kg dm), being the precursor of all hydrolyzable tannins [39]. In lower quantities, protocatechuic acid and p-hydroxybenzoic acids are also present [39, 40] (Figure 2).
Regarding hydroxycinnamic acids, they are principally located in skins, being p-coumaric, caffeic, ferulic, and sinapic acids the most significant. It should be reminded that p-coumaric and caffeic acids can be found esterified by the glucose of the anthocyanin monoglucosides forming their acylated derivates. In grapes (mainly white) and also in wines, hydroxycinnamic acids are mainly esterified with tartaric acid forming caftaric, p-coutaric, or fertaric acids (from caffeic, p-coumaric, and ferulic acids, respectively) [18] (Figure 2).
Phenolic acids, and overall, hydroxycinnamic acids can act as copigments. Indeed, they are implicated on the formation of new more stable pigments (pyranoanthocyanins) in wine and, in consequence, are considered as color stabilizer agents of young red wines, through copigmentation with anthocyanins [3]. Moreover, they are also associated with the sensory perception of astringency and bitterness [41].
Stilbenes (1,2-diphenylethylene) are formed from two phenyl rings linked together by an ethylene bridge generating a C6▬C2▬C6 structure. The aromatic rings are generally substituted by different functions such as hydroxyl, methyl, methoxyl, prenyl, or geranyl groups. Moreover, monomeric units (resveratrol) can also be coupled, leading to the formation of more complex stilbenes. Their composition and content are extremely variable depending on different biotic (attack of plant pathogens) and abiotic factors including grape variety and ripening stage [42].
In grape berries, stilbenes are mainly concentrated in skins [43]; only trace amounts are reported in seeds [44]. In addition, red varieties seem to present higher stilbene content than white ones [45]. The major stilbenes in grapes are the glucosides piceid (mean 1.3 mg/kg fresh weight (fw)), resveratrol (mean 1.1 mg/kg fw), and astringin (mean 0.5 mg/kg fw) [46] (Figure 2). Furthermore, other minor monomers were identified such as pterostilbene and isorhapontigenin [47]. Finally, different dimers as pallidol, ε-viniferin and δ-viniferin, trimers such as miyabenol C and α-viniferin, and tetramers as hopeaphenol and isohopeaphenol have also been reported [46] (Figure 2).
Oak wood is the preferred material for the manufacture of barrels, casks, or whatever derived wood product (chips, blocks, winewoods, tankstaves, etc.) used during fermentation and/or aging of wines. Resistance, flexibility, easy handling, and low permeability make oak wood particularly suitable for wine maturation and storage, in relation to mechanical, physical, and chemical properties provided by other woods [48].
Regardless of the species, oak heartwood is basically composed by cellulose, hemicelluloses, and lignin, representing approximately 90% of dry wood and acting as key structural polymers of wood matrix. The remaining 10% of dry wood corresponds to an extractable fraction, mainly consisting of phenolic compounds but also presenting low molecular weight compounds and volatile compounds. Lignans, coumarins, phenolic acids, and phenolic aldehydes may be found in the oak wood phenolic fraction, but hydrolyzable tannins are the major constituents [49].
Depending on the release of gallic or ellagic acid under acidic conditions, hydrolyzable tannins may be classified, respectively, as either gallotannins or ellagitannins [50]. Gallotannins are the simplest hydrolyzable tannins with a structure consisting of polygalloyl esters of glucose. The oxidative coupling of their galloyl groups converts gallotannins to the related ellagitannins [51].
Ellagitannins are the major nonvolatile extractives from oak heartwood (Figure 3). These oak phenolics have a specific structure, consisting of an glucose open-chain esterified at positions 4 and 6 by a hexahydroxydiphenoyl unit (HHDP) and a nonahydroxyterphenoyl unit (NHTP) esterified at positions 2, 3, and 5 with a C-glycosidic bond between the carbon of the glucose and position 2 of trihydroxyphenoyl unit [52]. Among ellagitannins, the monomers castalagin and vescalagin largely predominate in oak wood, representing from 40 to 65% by weight of total ellagitannins [53, 54, 55, 56, 57, 58] (Table 2). Six additional ellagitannins have been identified in oak wood: the lyxose/xylose-bearing monomers grandinin and roburin E, the dimers roburins A and D, and the lyxose/xylose-bearing dimers roburins B and C [64].
Chemical structure of main ellagitannins present in oak wood.
Treatment | Species | Monomers | Pentosylated monomers | Dimers | Pentosylated dimers | Total ellagitannins | References | ||||
---|---|---|---|---|---|---|---|---|---|---|---|
Castalagin | Vescalagin | Roburin E | Grandinin | Roburin A | Roburin D | Roburin B | Roburin C | ||||
Untreated wood | Quercus robur | 5.82–27.50 | 1.76–23.90 | 0.69–13.70 | 1.00–10.40 | 0.39–6.64 | 0.95–5.34 | 0.44–4.67 | 0.47–6.29 | 13.47–91.02 | [54, 56, 57, 58, 59] |
Quercus petraea | 1.63–11.76 | 1.05–8.01 | 0.78–4.44 | 0.76–5.47 | 0.13–2.05 | 0.17–3.27 | 0.29–2.41 | 0.06–2.25 | 5.87–34.41 | ||
Quercus alba | 1.19–5.29 | 0.70–2.83 | 0.29–1.38 | 0.38–2.17 | 0.31–0.70 | 0.08–0.67 | 0.13–0.83 | 0.04–0.54 | 3.48–13.14 | ||
Seasoned wood | Quercus robur | 1.43–13.00 | 0.85–10.60 | 0.77–9.20 | 0.36–6.15 | 0.09–1.91 | 0.18–2.52 | 0.09–0.48 | 0.14–1.72 | 3.93–43.97 | [53, 55, 56, 60, 61, 62] |
Quercus petraea | 0.90–15.91 | 0.39–11.76 | 0.39–7.98 | 0.12–5.80 | 0.05–2.73 | 0.02–2.58 | 0.04–1.84 | 0.01–1.97 | 1.98–39.77 | ||
Quercus alba | 0.41–4.45 | 0.15–6.44 | 0.15–2.30 | 0.07–1.81 | 0.03–0.40 | 0.02–0.84 | 0.02–0.79 | 0.01–0.20 | 0.89–8.93 | ||
Toasted wood | Quercus robur | 3.60–5.44 | 0.89–1.15 | 1.80–2.39 | 0.28–0.66 | 0.41–0.47 | 0.45–0.50 | 0.17–0.20 | 0.12–0.19 | 7.72–11.00 | [53, 60, 62, 63] |
Quercus petraea | 1.75–3.79 | 0.36–1.46 | 0.09–2.23 | 0.13–0.59 | 0.20–0.50 | 0.16–0.44 | 0.10–0.22 | 0.03–0.17 | 3.53–8.96 | ||
Quercus alba | 0.21–0.44 | 0.08–5.28 | 0.04 | 0.02 | ND | 0.04 | ND | 0.01 | 0.55–5.72 |
Ellagitannin concentration in untreated, seasoned, and toasted oak wood from the main Quercus species used in cooperage.
All values are displayed in mg/g of wood. ND, not detected.
Since ellagitannins are very soluble in wines and spirits, with a high reactivity, their levels in oak-aged beverages are much lower than what could be expected. When comparing both main monomers, vescalagin presents a more polar configuration that confers it a lower stability in hydro-alcoholic solutions [65]. From a sensory point of view, their level and profile may affect the astringency and bitterness of wine [66].
The level of ellagitannins in oak heartwood depends on the botanical species, the geographical origin, the single-tree variability, the sampling position in the tree, the grain, and the processing of wood in cooperage, notably the type and length of both seasoning and toasting periods.
Among the more of 150 oak species classified in the genus Quercus, the most frequently used in cooperage for winemaking are Quercus robur (pedunculate oak) and Quercus petraea (sessile oak), both growing in Europe, and Quercus alba, commonly known as American white oak, growing in the United States [5]. American oaks differ from European species not only because of their mechanical properties (higher density and resistance and lower porosity and permeability) [60] but also for the chemical composition of their phenolic fraction. Ellagitannin concentration is generally lower in Q . alba than European species, which in turn show a greater ellagitannin content in pedunculate oak than in sessile oak (Table 2) [53, 55, 56, 57, 58].
Until recently, French and American oak forests have been the quintessential source of wood for cooperage. Meanwhile, over the last few years, a huge number of studies on pedunculate and sessile oaks from different European origins (Hungary, Poland, Russia, Romania, Slovenia, Spain, Ukraine, and Moldova, among others) confirm their prospective use for maturation of quality wines [60]. Oaks from these new European origins appear to present ellagitannin concentrations halfway between French and American oaks [5, 58].
The width of oak wood rings (“grain”) is of great importance for the choice of oak wood for barrel and cask making, since it influences the wood chemical composition and affects the contribution of oak aging to wine quality. The higher the grain size, the larger the amount of ellagitannins released and the faster that release [67]. Furthermore, the grain size also exerts an effect on the oxygen transfer ratio (OTR): the smaller the grain size, the greater the OTR, and the faster the wine maturation [68].
Fresh wood cannot be directly used in winemaking, due to the great percentage of humidity (up to 70%), an excess of phenolic compounds, and a shortage of aromatic constituents. Oak wood conditioning in cooperage includes two stages that will determine the enological quality of wood. Both seasoning and toasting affect the structure and final chemical composition of the wood that is going to be in contact with wine.
Seasoning allows, not only reduction of humidity in wood, but also fiber contraction and wood maturation. During this process, an important decline of ellagitannin content is observed due to different physical, chemical, and biochemical mechanisms involved: stave leaching by rain, hydrolytic oxidative degradation, polymerization, and fungal enzymatic activity [56]. These phenomena occur particularly in the surface of wood and, in a lesser degree, but uniformly, in the inner wood [53]. Among the different oak wood seasoning methods, natural seasoning in open air seems to be more effective than mixed and artificial methods in reducing the excess of ellagitannins [56].
Toasting also induces an important modification of wood chemical composition, including an additional decline of the ellagitannin content. During toasting, castalagin is mainly oxidized in dehydrocastalagin, whereas its diastereomer vescalagin is reduced in deoxyvescalagin [69]. Similar deoxy- and dehydro-derivates have been observed for roburins A and D, respectively.
Ellagitannin degradation, and in turn their sensory impact on wines, may be modulated by changing the toasting thermal profile (temperature and length) [5]. In this sense, an ever-widening range of toasting levels is available at cooperages for all oak wood products. The higher the toasting level, the greater the ellagitannin decomposition, via dimerization and hydrolysis reactions, as well as formation of copolymers with cell-wall components [64].
Recently, new compounds that showed [M-H]-ion peak at m/z 1055.0631 (compound A) and at m/z 1011.0756 (compound B) have been identified as a result of thermodegradation of ellagitannins. The A compound corresponds to castacrenin E which is the oxidation product of castacrenin D, a vescalagin with an additional aromatic ring (gallic acid) to the C-1 through a C▬C bond. The levels of these compounds, found under experimental conditions and further searched in commercial oak wood, are dependent on both oak wood size and toasting method [70].
But, the extraction of oak phenolics into wine depends not only on the pool of potential extractible compounds originally present in wood, determined by the abovementioned factors, but also on the wine matrix, the aging length, and the exposed wood area to wine volume ratio.
During the grape ripening phase, the physiological and biochemical changes determine grape quality. The first period of grapes growth consists mostly of cell division and expansion, followed by a rapid growth phase during which the berry is formed and the seed embryos are produced. In this period, several compounds are accumulated in the berries, especially the tartaric and malic acids, conferring the acidity of the future wine. During the first growth period, several polyphenolic compounds increased like hydroxycinnamic acids in grapes’ pulp and skin and tannins and catechins in the skin and seed. The most important changes in grapes composition happen during the second growth phase (the ripening stage). Grapes switch from small, hard, and acidic berries to larger, softer, sweeter, less acidic, flavored, and colored ones. The majority of the solutes accumulated during the first growing phase remain at harvest. During the second period, the malic acid is metabolized and used as an energy source, its proportion decreasing toward the tartaric acid concentration, which remains almost unchanged. In general, the chemical composition of the final product is much more complex than in the raw material, due to the formation of new compounds [71].
Winemaking techniques involve the extraction of juice from ripe grapes, fermentation with yeast, and changes in polyphenolic composition that occur due to the participation of these compounds in various reactions such as copigmentation, cycloaddition, polymerization, and oxidation. These reactions begin after grape crushing, followed by fermentation and aging, contributing to the sensory properties of wines, mainly color and mouthfeel sensation. The total extractable phenolic content in grapes is encountered in seeds (60–70%), in the skin (28–35%), and in the pulp (about 10% or less). In the seeds, the phenolic content may range between 5 and 8%, by weight [72].
The understanding of the relationship between the quality of a particular wine and its phenolic composition remains one of the major challenges in enological research. For example, the anthocyanin fingerprints of varietal wines are proposed as an analytical tool for authenticity certification [27]. Patterns of some classes of flavonoids are under strict genetic control, and their distribution varies considerably among different grape cultivars [73, 74].
Several factors impact the wine phenolic composition, including the “terroir,” the grape variety and its degree of maturation before harvesting, or the winemaking process with its specific conditions of fermentation or aging [75]. Certain technological procedures, such as addition of sulfur dioxide (SO2) and/or ascorbic acid prior to crushing the grapes, maceration, yeast strain utilization and alcoholic fermentation, oxidation, or adsorption, can also influence the levels of phenolics during the winemaking process [76]. The addition of SO2 and pectolytic enzymes before fermentation caused an increase in color intensity, color stability, total phenolics, anthocyanins, catechin, and epicatechin in a red Italian wine [77].
In white grape musts, the predominant phenolic compounds are hydroxycinnamic tartaric acid esters as catechins and proanthocyanidins which are found mainly in their skins. The must fermentation of red wines is realized in the presence of both grape skins and seeds. During this process, phenolic compounds such as anthocyanins are subjected to various reactions, such as enzymatic oxidation, nucleophilic substitution, degradation, and cycloaddition of the carbonyl compounds leading to the formation of vitisins (A and B). These pyranoanthocyanins in red wine are mainly orange pigments. Moreover, the red wine color evolution and stabilization are mainly induced by the formation of polymerized pigments. The acidic hydrolysis of proanthocyanidins leads to the formation of flavan-3-ol unit or tannin oligomers with a carbocation in C4 position which can be attacks by positions C6 and C8 of another proanthocyanidins or an anthocyanin. This reaction will induce the modification of the condensed tannin polymerization degree or the formation of the polymerized pigment. These newly formed purple pigments induce the color modification of the young red wine. Moreover, polymerization through acetaldehyde between two condensed tannins or between condensed tannins and anthocyanins also occurs during the winemaking process and aging. The formation of these ethyl bridge compounds will also produce modification in the organoleptic properties of the wine and the color stabilization since the ethyl-bridged anthocyanin-tannin compounds also exhibit purple color [72]. During the red wine maturation in bottles, all these newly formed purple polymerized pigments will undergo slight oxidative reaction to slowly form some more orange pigments which together with pyranoanthocyanins are forming the color of old wine.
The winemaking process involves the alcoholic fermentation of must, often followed by malolactic fermentation (MLF). When MLF is completed, the wine is subjected to different clarification and stabilization treatments and/or is stored in oak barrels for aging for a variable period of time. MLF and aging in oak barrels are two enological processes which modify the composition and sensory characteristics of the wines [5, 78, 79, 80]. When oak wood derivatives like chips are added after fermentation, wines seem to have a greater aging potential compared to the wines fermented with chips due to their higher ellagitannin content and enhanced condensation reactions. On the other hand, color stabilization and tannin polymerization occur faster when chips are added during fermentation, resulting in shorter aging periods suitable for early consumed wines [81]. MLF in tanks may simplify the control of the process; however, the use of oak wood during the MLF stage affects the chemical and sensory attributes of wines. In red wines, MLF container plays an important role on proanthocyanidin and anthocyanin concentration and evolution as oxygen in small quantities favors polymerization reactions among anthocyanins and tannins. Wines performing MLF in tanks present a higher total proanthocyanidin concentration (5.8 g/L wine) than that of those which accomplished MLF in medium-toasted barrels (4.9 g/L wine). The major wine glucosidic anthocyanin, malvidin, showed as well greater levels in wines carrying out their MLF (33 and 26 mg/L wine, respectively, for tank and barrel MLF). Regarding ellagitannin concentration, their content is strongly influenced by both barrel toasting and MLF container. For instance, in the case of medium-toasting barrels, castalagin was found at concentrations twofold times higher (19 mg/L wine) when MLF was performed in barrels [79]. Concerning sensory results, the MLF strengthens the organoleptic preference of wine when it takes place in barrels [79, 80, 82]. In white wines, total ellagitannin concentration varied from 7.8 to 17.4 mg castalagin equivalents/L for wines performing MLF in tanks and barrels, respectively [83].
The phenolic composition of wine changes along the wine aging process reflects in the color and astringency level of the final product. From 1978 to 2005 vintage for Cabernet Sauvignon wine, phenolic compounds, total tannins, and total anthocyanins varied from 1735 to 2903 mg/L, from 1.3 to 2.2 g/L, and from 15 to 123 mg/L, respectively [29]. In general, the relative anthocyanin content decreases upon aging although this chemical modification is associated with a very clear change in color. This characteristic is often used as a quality standard for aged wines. One of the main factors responsible for anthocyanin loss is the storage temperature [84]. The majority of red wines aged are in contact with oak wood, whether in form of barrels or in form of oak wood derivatives. As a consequence, their phenolic composition changes due to the addition of oak wood extracted compounds. These compounds include hydrolyzable tannins (C-glucosidic ellagitannins), aromatic carboxylic acids, and several aldehydes. Regarding wine-air interactions, barrel structure allows a controlled entrance of oxygen, which is essential to the polymerization and the slight oxidative reactions between different types of flavonoids, leading to a modification of the organoleptic properties of the wine. Indeed, wood can affect wine composition and, consequently, organoleptic properties through different mechanisms. On the one hand, wine compounds can be adsorbed onto wood surface. On the other hand, compounds, such as ellagitannins, can be extracted from wood to the wine due to the hydro-alcoholic nature of the latter. Ellagitannins can take part in oxidation reactions that may favor the polymerization reactions between flavanols and between flavanols and anthocyanins. Furthermore, they can directly react with these types of compounds giving rise to flavano-ellagitannins or anthocyano-ellagitannins [85]. The formation of flavano-ellagitannins and the β-1-O-ethylvescalagin in red wines aged in oak barrels has been reported. The ellagitannin concentrations fluctuated between 4 and 8 mg/L, being castalagin the ellagitannin with the highest concentration, followed by mongolicain A [86]. As a consequence, ellagitannins can modulate wine astringency and color through interactions with these compounds. Strong correlations have been observed between ellagitannin concentration and both antioxidant capacity and astringency sensation [5, 63, 78]. The amount of ellagitannins released into the wine depends on the content in the oak wood barrel, which in turn is dependent on several factors (Section 3). For instance, after 12-month aging with woods, the total ellagitannin level revealed a large diversity of concentrations ranging from 6.3 to 26.1 mg of ellagic acid/L wine. The wine with heavy toast woods and the wine with low toast woods presented, respectively, the less and the highest ellagitannin concentrations [78]. Storage with oak can also cause a decrease in anthocyanins, catechin, and epicatechin but an increase in total phenolic content and a stabilizing effect on color [77].
Besides the winemaking process, and oak wood aging, wine can be further exposed to oxygen during aging in the bottle, depending on the oxygen permeability of the closure. Because of the extremely low rates of oxygen ingress through a closure, this form of oxygen exposure has been referred to as nano-oxygenation. Oxygen transmission rates (OTR) of wine closures may vary widely depending on closure type and strongly influence the evolution of white and red phenolic composition and astringency during bottle aging [87].
“In-mouth” sensory properties of red wines encompass multiple interacting sensations such as acidity, sweetness, bitterness, retronasal aroma perception (flavor), viscosity, warmth, and astringency. Among these, the sensation of astringency and the taste of bitterness are related to phenolic compounds.
Bitterness perception is a taste recognition mediated by taste buds present in the tongue papillae. Each taste bud, consisting of approximately 50–100 taste receptor cells, is innervated by multiple taste fibers that transmit nervous signals to the brain [88]. In humans, each bitter receptor cell contains approximately 25 bitter G protein-coupled receptors encoded by a TAS2R gene family. It was evidenced by Soares and colleagues [89] that different phenolic compounds activate distinguished combination of TAS2Rs: epicatechin stimulated three receptors whereas procyanidin trimer only one. In general, in wines containing a large number of polyphenols, the taste of bitterness is attributed to flavan-3-ols and their polymers, although it is also able to be elicited by some flavonols [90], hydroxycinnamates, and benzoic acid derivatives [91].
While bitterness is a gustatory sense recognized by nervous signals, astringency is an oral sensation involving dryness and puckering [92]. According to the American Chemical Society, astringency refers to “the complex of sensations due to shrinking, drawing or puckering of the epithelium as a result of exposure to substances such as alums or tannins” [93]. It has been classically postulated that tannins possess the ability to interact with salivary proteins, with or without precipitation of the corresponding complexes [94]. In fact, the name of “tannins,” originating from “tanning” of leather, has been used over decades for their capability of binding with proteins. The mechanisms of tannin-protein interactions involved different types of interaction such as the hydrophobic interactions, which are the predominant mechanisms involving the hydrophobic nature of the condensed tannin carbon skeleton and the apolar regions of the proteins. Together with hydrophobic interactions, some hydrogen interactions also occur between the carbonyl function of proline and the hydroxyl functions of phenols as well as some ionic interactions. This mechanism and thus the final astringency of wine are influenced by many factors, such as the structure and amount of condensed tannins in the wine as well as the composition of the wine matrix [95]. Indeed, the intensity and the quality of the astringency perception are influenced by the concentration of condensed tannins [96], their degree of polymerization [97], their percentage of galloylation [98], and prodelphinidins [99]. The matrix, on the other hand, impacts the perception according to its acidity [100], its ethanol concentration [101], and the presence of macromolecules such as polysaccharides. Fontoin and co-workers demonstrated that the astringency sensation of grape-seed oligomer tannins decreased with increasing pH and the percentage of ethanol [100]. For example, Cabernet Sauvignon wines having a mDP between 2.0 and 4.0 were characterized as mellow and slight astringent. Meanwhile, a rougher sensation (tannic) was perceived for wines with a mDP higher than 4.0. [102]. The analytical determination of the proanthocyanidin content and the type of subunit that is dominant in tannin chains might be a valuable tool for astringency estimation during wine aging [103]. Astringency intensity is influenced by the source of proanthocyanidin (seed or skin) and by well-defined proanthocyanidin fractions (oligomeric or polymeric). Polymeric seed fraction was perceived more astringent than the monomeric/oligomeric one, whereas polymeric skin fraction was characterized less tannic than the monomeric/oligomeric skin fraction, indicating a negative relationship between astringency intensity and % of prodelphinidins [104]. The presence of epigallocatechin units in the proanthocyanidins has been shown to lower the “coarse” perception through the increase of the degree of B-ring trihydroxylation. Furthermore, seed fraction with a higher proportion of galloyl group and a lower mDP was perceived to be bitterer than the skin fraction [105]. Both the interflavanoid bonds and stereochemistry of subunits impact bitterness sensation: catechin-(4–6)-catechin dimer was bitterer than both catechin-(4–8)-catechin and catechin-(4–8)-epicatechin [91].
Moreover, astringency sensation perceived always reduces with the increasing saliva volume flow rate. A linear correlation was found between protein concentration and tannin-binding affinity. The saliva proteins, including PRP family (acidic, basic, and glycosylated), α-amylase, statherin, histatins, and mucins, show diversified ability to interact with tannins [106]. Some proteins are specifically bound to astringents. For instance, tannins and alum precipitated PRPs, while acid and alum precipitated mucins [107, 108].
Ellagitannins (hydrolyzable tannins) impart an oral sensation described as astringent at relatively low threshold concentrations spanning from 0.2 to 6.3 mmols by means of the half-tongue test in bottled water (pH 4.5). Due to their lower astringent taste thresholds, hydrolyzable tannins are usually perceived as more astringent than condensed tannins (1.1 μM for both castalagin and vescalagin, compared to 410 μM for catechin, 930 μM for epicatechin, 240 μM for procyanidin B1, 190 μM for procyanidin B2, and 200 μM for procyanidin B3) [66, 69, 109]. Among ellagitannins, the monomers grandinin and roburin E exhibited an astringent mouthfeel at the lowest taste thresholds (0.2 μM), whereas values for dimers ranged between 2.9 and 6.3 μM. Thus, the C-glycation of castalagin and vescalagin seems to favor the astringent sensation, while dimerization seems to reduce it [66]. When the same concentration of ellagitannins and skin and seed tannins was tested, the ellagitannins were perceived softer [104].
Interaction of ellagitannins with salivary proteins has been poorly investigated up to now, probably because of their lower wine content compared to condensed tannins. Even if perceived as more astringent, ellagitannins have been noted as poorer protein precipitants than condensed tannins [94]. Soares and co-workers [110] stated that ellagitannins act as multidentate ligands cross-linking different salivary protein units, via their galloyl moieties. It is noteworthy to mention that these units are responsible for the antioxidant ability of hydrolyzable tannins; thus when complexed with salivary proteins, the antioxidant capacity of ellagitannins may be significantly impaired. At higher concentration levels, the main eight oak ellagitannins have also been observed to provide the wine with a bitter taste [66].
Apart from tannins, other polyphenolic compounds present in wine have been related with the overall perception of astringency sensation or bitterness. Very recently, some works have provided evidence about the interaction of anthocyanins and pyranoanthocyanins with salivary proteins. Indeed, malvidin-3-O-glucoside, the major anthocyanin of wine, has demonstrated to interact with acidic proline-rich proteins (aPRPs) showing dissociation constants (KD) calculated by NMR of 1.88 mM [111] that can be compared to those obtained for procyanidins (dimers B1–4 and trimer C2) (between 0.4 and 8 mM) [112]. In addition, Paissoni and colleagues [113] tested the interaction with saliva proteins of the three representative of wine anthocyanins (glucosides, acetylated, and cinnamoylated) proving that cinnamoylated anthocyanins are the most reactive and also those that present the lowest perception threshold in wine model solutions. More recently, another work showed that pyranoanthocyanins (pyranomalvidin-3-glucoside, pyranolmalvidin-3-glucoside-catechol, and pyranomalvidin-3-glucoside-epicatechin) can also able to interact aPRPs with KD even lower (more affinity) than for anthocyanins (between 0.87 and 1.73 mM) [114].
Concerning bitter taste, malvidin-3-O-glucoside has also demonstrated to stimulate one member of the bitterness receptor family (TAS2R7) at micromolar levels (12.6 μM) [89]. With regard to flavanols, the addition of quercetin-3-O-glucoside (0.25–2 g/L) to white and red wines resulted in a noticeable increase of astringency and bitterness evaluated by sensory analysis. In general, wines were described as smooth-tasting before the flavonol addition and became more astringent, rough, green, dry, bitter, and persistent in presence of quercetin-3-O-glucoside [33].
There is no doubt that wine is an extremely complex medium and that polyphenolic compounds play an essential role on its final sensory properties. There is an inestimable chemical diversity of polyphenols in both grapes and wines. Each family can be present in free or conjugated forms, with different hydroxylation levels and substitutions or even forming adducts between them. Starting with the raw material, phenolic compounds in grapes can vary substantially depending on several factors as ripening, viticulture practices, environmental conditions, and varietal or genetic differences. Such is the case that each cultivar may be considered as “exclusive” and consequently, the resulting wine too. Anthocyanins (mainly present in skins) and flavan-3-ols and condensed tannins (mainly present in skins and seeds) are the most abundant polyphenols in grapes. On the one hand, anthocyanins are responsible of the color of red wines, and their profile can be used as an analytical tool for authenticity certification. On the other hand, flavan-3-ols and condensed tannins are key compounds due to their implication on color stabilization and astringent and bitter properties. Finally, other phenolics as flavonols and hydroxycinnamic acids are mainly known for acting as copigments. Oak wood is commonly used during fermentation and/or aging of wines. The phenolic composition of oak wood will vary depending on species, geographical origin, and grain or wood processing. In quantitative terms, ellagitannins are the major phenolic constituents of oak wood, and their level and profile may affect the astringency and bitterness of wine. Winemaking produces important changes in polyphenolic composition. In fact, phenolics participate in several reactions such as copigmentation, cycloaddition, polymerization, and oxidation. Thus, new compounds as vitisins, ethyl-bridged anthocyanin-flavanol derivatives, or pyroanthocyanins are formed. Furthermore, wine aging in contact with oak wood affects the degree of complexity of phenolic compounds. In this sense, ellagitannins are actively engaged in oxidation reactions that favor the polymerization between flavanols and between flavanols and anthocyanins.
Overwhelming evidence has demonstrated that tannins (mostly condensed tannins), thanks to their ability to precipitate salivary proteins, are implicated on wine astringency. Astringency intensity, even if it is a multifaceted sensation complicated by a number of variables, is more influenced by the source of proanthocyanidin (seed or skin) and by well-defined proanthocyanidin fractions (oligomeric or polymeric). Up to now, ellagitannins’ direct impact on astringency and bitterness sensation remains still unknown. One of the major limitations of the half-tongue test used to evaluate their sensory impact is the absence of contact between the ellagitannins and the entire oral cavity. Further studies are needed under wine conditions. Additionally, recent studies highlight that other phenolic compounds such as anthocyanins/pyranoanthocyanins or flavanols may also interact with salivary proteins and bitterness receptors. Thus, a new research line in the field of sensory properties linked to wine phenolic compounds sensory properties is opened.
Pandemics and epidemics of infectious origin are large-scale outbreaks that can greatly increase morbidity and mortality globally or over a wide geographic area, respectively [1]. Pandemics have occurred throughout history and appear to be increasing in frequency in the last centuries. Noteworthy examples include the Black Death at the end of the Middle Ages, Spanish flu in 1918, the 2014 West Africa Ebola epidemic or the current COVID-19 pandemic. The direct impact of pandemics on health can be dramatic. These large outbreaks can disproportionally affect younger or active workers, but vulnerable populations such as the elderly are at a particular high-risk. Pandemics can cause acute, short-term as well as longer-term damage to economic growth due to public health efforts, health system expenditures, and aid to affected sectors. Evidence suggests that epidemics and pandemics can have significant social and political consequences too, by debilitating institutions, amplifying political tensions, stigmatizing minority populations, or encouraging sharp differences between social classes [2].
Outbreaks by respiratory ribonucleic acid (RNA) viruses such as influenza or coronaviruses entail the principal threat due to their ease of spreading among humans, their potential severity and recurrence. However, other RNA viruses such as flaviviruses (Zika) or filoviruses (Ebola) must be taken into consideration due to a great overall burden of morbidity and mortality [3]. Antiviral drugs can help mitigate a viral outbreak by reducing the disease in infected patients or their infectiousness. While these drugs can be very successful against some viruses (e.g. hepatitis C virus [HCV]) [4], they are not universally effective as exemplified in the current SARS-CoV-2 pandemic [5]. Nowadays, having effective vaccines may be the only tool to reduce susceptibility to infection and thus, prevent the rate of virus spread [2].
Vaccination has dramatically decreased the burden of infectious diseases. Vaccines have saved hundreds of millions of lives over the years [6]. It has been estimated that approximately 103 million cases of childhood diseases were prevented in the United States through vaccination between 1924 and 2010 [7]. The eradication of smallpox in 1980 through vaccination is considered one of the crown accomplishments of medicine. Despite these achievements, effective vaccines have been developed against just over 30 pathogens among bacteria and viruses. There are many pathogens, including viruses such as human immunodeficiency virus (HIV) or respiratory syncytial virus (RSV), for which all efforts for vaccine development have failed so far. In addition, current available vaccines for worldwide important viral diseases like influenza are suboptimal, especially in the elderly, resulting in vulnerability among billions of at-risk populations [6]. On the other hand, having a new effective and safe vaccine in time to control highly contagious emerging viruses that cause epidemic or pandemic threats is an almost impossible task considering the timeframes for vaccine development. This includes preclinical and clinical research, its approval by the regulatory authorities, as well as its production and distribution [3].
Altogether, it has been postulated that one possibility of filling the gap between the appearance of a viral outbreak by an emerging pathogen and the availability of a specific vaccine is to take advantage of the heterologous protection of some existing vaccines, in order to increase the non-specific resistance of the host through trained immunity [8, 9].
Conventional (specific) anti-infectious vaccines are biological preparations containing live-attenuated or dead microorganisms, their antigens or nucleic acids encoding for them, designed for specific pathogens. The purpose of vaccination is to induce a long lasting adaptive immune response against key antigens able to confer host resistance for future encounters with the corresponding pathogen. Either the production of antibodies, generation of T helper/effector cells, or both, may play a critical role in such a resistance, which greatly depends on the type of pathogen, the route of entrance and the host-pathogen relationship (e.g., extracellular and/or intracellular) [10]. Successful vaccines are highly effective not only in inducing long-lasting immunity against disease-causing pathogens, but also in providing herd immunity to the community that substantially restricts the spread of infection [6].
Most of the vaccines available today have been developed empirically and used successfully long before their mechanism of action on the immune system was understood. Early protection is associated to induction of antigen-specific antibodies, being their quality (avidity, specificity, or neutralizing capacity) key factors for their efficacy. Long-term protection relies on the persistence of vaccine antibodies and availability of immune memory cells capable of rapid and effective reactivation with subsequent microbial exposure. On the other hand, T cells have a critical role in the induction of high affinity antibodies and immune memory. Furthermore, T cells have a direct role in protection conferred by some vaccines, including the tuberculosis Bacille Calmette-Guérin (BCG) vaccine [11].
Vaccines using whole pathogens have been classically classified as either live attenuated or inactivated (killed). Subunit vaccines contain just selected antigens (e.g., proteins, polysaccharides). Recently, due to a growing availability of bioinformatics and sequencing tools, there has been an increase interest on so-called “rational” vaccine design approaches for subunit vaccines, such as the reverse vaccinology [12]. In this regard, modern vaccines include recombinant proteins or nucleic acids [13]. Rather than administering the antigen itself, DNA and mRNA vaccines targeting dendritic cells (DCs) encode the antigen of interest that will be produced by the vaccinated host, representing a new era in vaccinology [14]. In fact, the first RNA vaccine licensed for humans in Western countries has been recently developed for SARS-CoV-2.
As commented before, a vaccine response is linked to the induction of T and B cell specific responses to the antigens contained in the vaccine. This requires lymphocyte activation, proliferation and differentiation on specialized lymphoid tissues (e.g lymph nodes), where antigen presenting cells, like DCs for T cells or follicular dendritic cells (FDCs) for B cells, are present. Mature DCs are recruited into the T cell areas of lymph nodes from the periphery, e.g., at the site of injection of the vaccine. DCs express pattern recognition receptors (PRR) that recognize evolutionary conserved pathogen-associated molecular patterns (PAMPs) that are not contained in self-antigens and are identified as “danger signals” [15]. When immature DCs are exposed to the vaccine-derived antigens at the site of vaccination, they uptake them and become activated [16]. This activation will lead to their maturation with the expression of homing receptors at their surface, triggering DC migration to the draining lymph node through afferent lymphatic vessels, where the activation of T and B lymphocytes will occur. Mature DCs process the up-taken antigens and present them to naïve T cells associated to molecules of the major histocompatibility complex (MHC) within the T cell areas of lymph nodes. On the other hand, unprocessed native antigens, either free or complexed with antibodies or complement, access the B cell areas of lymph nodes (lymphoid follicles) where they are captured by FDCs and displayed from their cell surface to the B cells. Antigen-specific B cells will rapidly proliferate forming a germinal center and differentiate into plasma cells producing low-affinity immunoglobulin (Ig) M antibodies. The B cells will then receive additional signals from activated T cells, undergoing isotype antibody switch from IgM to IgG or IgA and affinity maturation of the antibodies produced.
For a vaccine to be immunogenic enough, DC activation, that can be achieved by adjuvants, is essential. Live attenuated and inactivated whole-cell vaccines are considered “self-adjuvanted” as they naturally present sufficient PAMPs to activate innate immune cells, including DCs; thus, promoting a robust antigen-specific immune response. In contrast, subunit vaccines generally require different types of adjuvants to enhance and/or drive the immune response in the desired direction [15, 17].
Viral outbreaks appear when there is a sufficient number of susceptible individuals within a nearby population. Although susceptibility is a balance between host factors (high/low resistance) and pathogens (high/low virulence), in many cases it reflects a lack of prior contact with a given pathogen. In general, this is related to the emergence of new viruses or the lack of effective vaccines against known viruses. As pointed above, the development of effective vaccines is not an easy task against certain viruses. We are still lacking vaccines for some of the most lethal viral infections, including HIV and MERS-CoV, among others. These pathogens are difficult to tackle, as we do not fully understand their mechanisms to evade the immune system or how to elicit protective immunity against them [13]. However, encouraging progress is being made against these pathogens and there are currently several “pipeline vaccines” in development, such as RSV, universal influenza vaccine, and SARS-CoV-2 [18, 19, 20]. Apart of SARS-CoV-2 for obvious reasons in the current pandemic, there is an urgency to have a universal influenza vaccine that provides a broad and durable protection from influenza virus infection. Yet, the high level of antigenic diversity and variability, and antigenic drift in the surface antigens, enable these viruses to escape antibody-mediating neutralization [21]. On the other hand, there is a number of vaccines currently licensed, including the influenza A virus vaccine, that provide incomplete protection, especially in high-risk groups [22]. Mumps outbreaks observed in Ireland, United Kingdom and United States in vaccinated subjects with Measles Mumps Rubella (MMR) vaccine is another example [23]. Different factors have been postulated to contribute to mumps outbreak, including waning immunity and primary and secondary vaccine failure. Yet, their actual contribution is not fully understood [23].
Vaccine efficacy must consider different target populations as well. Adaptive immune response to vaccines may be limited in newborn and the elderly. Early in life, immune responses are dampened compared to adults [24, 25]. Neonates have underdeveloped germinal centers in lymph nodes and the spleen, and low expression of B cell receptors which in turn results in low levels of primary IgG responses to infections and vaccines [26]. As we age, our immune system undergoes age-related changes that lead to progressive deterioration of the innate and the adaptive immune responses, this is termed immunosenescence. The most common features of immunosenescence are short-lived memory responses, impaired response to new antigens, increased predisposition to autoimmune diseases and low-grade systemic inflammation (inflammaging) [27, 28]. Immunosenesence results in increased susceptibility to infections and deficient response to vaccination causing high hospitalization and mortality rates. For example, influenza vaccine efficiency has been reported to be 17–53% in the elderly, compared with the 70–90% efficacy in young adults [29]; and vaccination with Varicella zoster virus (VZV), also an important pathogen in elderly people, only partially prevents reactivation of herpes zoster [27].
If the difficulties listed above are outlined for existing or developing vaccines, quickly obtaining an effective vaccine to urgently control a new virus outbreak is almost an impossible task in the short-term as pointed above. This is well exemplified by the SARS-CoV-2 vaccine race pushed by the devastating COVID-19, with more than 100 vaccine candidates in the running. It is considered that no less than 1 year will last the time until the first licensed vaccine can provide protection in the best scenario [30]. This, in spite of greatly shortening the usual clinical development time and regulatory obstacles for a new vaccine and, therefore, without knowing its true performance and/or safety in the medium term compared to other authorized vaccines [31].
It has become evident from epidemiological, clinical and experimental data that some conventional whole-cell vaccines, like BCG and others, also provide resistance to infectious diseases not related with the specific pathogen targeted by the vaccine [32, 33, 34]. Much of these non-specific “heterologous” effects appear to depend on the activation of innate immune cells by the PAMPs contained naturally in these vaccines [10], although other mechanisms such as cross-reactive epitopes between different pathogens could also account for this protection in some cases [35].
Immunological memory, understood as the ability to “remember” past encounters with pathogens, has been classically attributed to the adaptive branch of the immune system exclusively, by virtue of the antigen-driven clonal expansion of T and B lymphocytes and exemplified by the mechanism of conventional specific vaccines pointed above. However, the notion that innate immunity was unable to induce immunological memory has been challenged in recent years, particularly from studies in organisms that lack adaptive immunity, such as plants or invertebrates, as well as early studies in mice lacking the adaptive immune system [8, 36]. Altogether, the term ‘trained immunity’ was coined to define an innate immune memory that lead the innate immune system to an enhanced response to secondary challenges [37]. Importantly, trained immunity seems to be underlying the heterologous effects of an increasing number of vaccines [38, 39, 40].
What is trained immunity? - Trained immunity is defined as the memory of the innate immune system, where an encounter with a first stimulus (e.g. a microbial insult) results in a subsequent long-term adaptation and enhanced non-specific response by innate immune cells against a secondary challenge (the same or unrelated), thus providing non-specific, broad-spectrum, long-term protection in case of infection [8, 9, 37, 41].
Which cells can be trained? - Trained immunity properties have been defined for distinct cell subsets of the innate immune system [9, 42], including natural killer (NK) cells and innate lymphoid cells [43]. Of note, training of myeloid cells [42], particularly monocytes and macrophages [44, 45], and more recently DCs [46, 47] and hematopoietic stem cells [48], have been extensively studied. Finally, the acquisition of this immunological memory has also been demonstrated to a lesser extent for non-immune cells [49].
How to get trained? - A wide variety of stimuli can train innate immune cells, particularly when considering monocytes and macrophages [9, 50]. Among infectious agents, live microorganisms such as the tuberculosis vaccine BCG [51], Candida spp [52] or viruses [53, 54]; bacterial components, such as flagellin, lipopolysaccharide, muramyl dipeptide [55], fungal components as β-glucan [52] or even helminth products [56]. In general, microbial ligands engaging some PRR, like C-type lectin receptors (CLRs), nucleotide-binding oligomerization domain-like receptors (NLRs) are well established training inducers, whereas those engaging toll-like receptors (TLRs) may have opposite effects depending on the TLR type and concentration [55, 57]. Intriguingly, not only infectious agents but also endogenous inducers and metabolites such as oxidized low-density lipoprotein or mevalonate can induce trained immunity [50].
What hallmarks define trained immunity? - In contrast to adaptive immune responses, epigenetic reprogramming of transcriptional pathways — rather than gene recombination — mediates trained immunity. This training phenomenon comprises three key hallmarks that occur at the intracellular level: increased cytokine production upon rechallenge, changes in the metabolism and epigenetic reprogramming [9, 58, 59], which eventually support increased protection upon infection.
Among those cytokines whose production is augmented after re-exposure in trained cells, proinflammatory molecules such as tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-1β and interferon γ (IFN-γ) are fairly constant [45, 52, 55, 60, 61]. Modulation of IL-10 varies between studies [45, 52, 56, 62, 63]. A noted shift from oxidative phosphorylation to aerobic glycolysis (Warburg effect) is the main change in cellular metabolism during the induction trained immunity [64]. Moreover, glutaminolysis, cholesterol synthesis and the tricarboxylic acid cycle are non-redundant pathways required for trained immunity to take place [64, 65]. Epigenetic reprogramming, mainly mediated by histone modifications, is one of the bases for the long-lasting effect of trained immunity [8, 66, 67, 68]. Immune pathway activation and changes in metabolism serve as basis for epigenetic rewiring [65]. As a result, epigenetic modifications have been found at the level of important promoters for the training process, which makes chromatin more accessible and conditions gene expression patterns of trained cells upon stimulation with a secondary challenge [69].
As a result of the whole process, enhanced, broad-spectrum, non-specific protection mediated by innate immune cells is found upon infection. This cross-protection has been observed for a wide range of human pathogens including fungi [51, 52], parasites [70, 71] and different bacterial infections [72, 73, 74, 75]. Importantly, induction of trained immunity has been proved to be effective against viral infections including yellow fever [76], influenza A virus [77] and others [78, 79]. In this line, the induction of this phenomenon has been also proposed as a tool for reducing susceptibility to emergent SARS-CoV-2 infection, as will be described at the end of the chapter [78, 80].
How long does trained immunity last? – Trained immunity phenotypes have been observed for months and up to one year after the training insult. This was initially controversial, as trained immunity properties had been attributed to short-lived myeloid cells such as monocytes or DCs [38]. In this regard, several studies have shown that modulation of bone marrow progenitors is also an integral component of trained immunity, supporting the long-lasting effect of this phenomenon [9, 81]. In this way, trained immunity inducers [82, 83, 84, 85] would be able to reprogram and induce expansion of hematopoietic progenitors with a particular bias to the myeloid lineage. Thus, bone marrow-derived mature cells would be also trained [86], showing improved clearance of infection [83].
Complementary to progenitor reprogramming, peripheral trained immunity induction would take place in tissue-resident cells [9]. This is especially relevant at the mucosal level, where cells encounter most of the infectious training inducers. Alveolar macrophage (AM) memory was demonstrated following viral infection [87, 88]. Training of these long-living cells led to increase antimicrobial properties, independently of systemic immunity [87, 89]. This local training of AM was further reproduced following respiratory mucosal administration of tuberculosis vaccine, being crucial for Mycobacterium tuberculosis clearance [90]. On the other hand, training of NK cells lead to long-lived, self-renewing, stable expanded cells with memory-like properties, both in an antigen-dependent or independent manner [91, 92, 93]. Finally, it was also reported that self-renewing long-living skin epithelial stem cells exhibited local trained immunity, providing faster wound healing in primed mice than in naïve mice [94, 95].
Non-specific effects of vaccines have been extensively studied and reported over the last decades. Although trained innate cells could partially account for these effects, involvement of adaptive immunity has also been suggested [96]. An adaptive immune mechanism of non-specific effects could be heterologous immunity; vaccine antigens can give rise to T cell cross-reactivity against other antigens that may confer some protection against unrelated pathogens [96, 97].
However, innate immune cells constitute the bridge between the intrusion of microbial threats and the activation of adaptive immunity. As said before, following sensing of pathogens by PRRs, activated innate immune cells secrete different factors and act as antigen-presenting cells (APCs) to initiate activation of adaptive immunity [98]. Thus, it would not be unexpected that trained innate immune cells, within their acquired enhanced properties, would be able to induce stronger adaptive immune responses [39]. In this regard, BCG vaccine, a well-known trained immunity inducer, has shown to enhance the antibody titer and alter heterologous T cell responses against a wide range of vaccines and unrelated infections [99, 100, 101]. In different experimental models, BCG-mediated protection against viral and Plasmodium infections was abrogated in the absence of T cells. In these models, BCG vaccination has been mainly associated with modulation of CD4+ T helper (Th) 1 responses. Similar observations have been found in different clinical studies [99]. Of note, BCG vaccinated human volunteers displayed a long-lasting heterologous Th1 and Th17 response upon stimulation with unrelated pathogens and TLR-ligands [38]. To some extent, similar observations have been found in other vaccines such as diphtheria-tetanus-pertussis (DTP) or measles vaccine [99].
As said before, trained immunity properties have been recently described also for DCs. As being the most professional APCs, they emerge as crucial bridge for potentiating adaptive immune responses. In this sense, DCs with high immunostimulatory properties that enhance adaptive immune responses via IL-1β release had been described [102]. More recently, programmed memory DCs have shown to increase Th1/Th17 immunity and confer protection during cryptococcosis [46]. Finally, different polybacterial preparations of whole-cell inactivated bacteria, have shown to prime DCs and induce enhanced Th1, Th17 and IL-10 T cell responses against related and unrelated stimuli [103, 104]. This capability of modulating heterologous T cell responses by APCs have been also described to suppress pathogenic T cell immunity in experimental models of autoimmune encephalomyelitis [56].
As noted above, a hallmark of trained innate immune cells is the enhancement of some effector functions leading to increased non-specific resistance against a variety of pathogens. In this regard, β-glucan-trained monocytes show enhanced candidacidal activity and efficiently inhibit the C. albicans outgrowth [52]. Production of reactive oxygen species (ROS) has shown to be also affected by the induction of training. Thus, BCG-trained monocytes [45], β-glucan-trained macrophages [105] or β-glucan-trained neutrophils [106] produced increased amount of ROS following different challenges. Finally, increased phagocytosis and production of microbicidal molecules have been observed in β-glucan-trained macrophages [70, 105]. Mechanisms underlying this enhanced effector function could be an intrinsic cell reprogramming as consequence of the training, as well as be supported increased expression of different PRRs and surface molecules [45, 60, 87]. Altogether, these enhanced effector responses could improve pathogen clearance by increasing host resistance.
On the other hand, a substantial part of the adaptive immune response is directed at recruiting other effector cells from the innate immune system to eventually resolve an infection. Both T helper and B responding cells release cytokines, antibodies, and other mediators that activate monocytes, macrophages, NK cells or neutrophils to clear extracellular and intracellular pathogens [107]. Multiple studies have demonstrated the importance of IFN-γ-mediated priming in the activation of macrophages [108, 109], produced by CD4+ Th1 and CD8+ T cells [107]. In this sense, it has been previously demonstrated that adaptive T cells render innate macrophage memory via IFN-γ-dependent priming [87, 89]. Furthermore, a deep crosstalk between Th17 and neutrophils have been widely demonstrated, via production of IL-17 and other related cytokines [110].
Taken into account the potential role of trained innate cells in both the induction of adaptive and effector responses, a notable amplification loop in the global immune response could be considered (Figure 1).
Effect of trained immunity on ongoing immune responses. Induction of trained immunity allows trained cells to enhance adaptive immune responses and vice versa, final effector functions of trained cells can be further potentiated by enhanced adaptive responses.
Based on trained immunity pillars, a next generation of anti-infectious vaccines has been postulated, coined as ‘Trained Immunity-based Vaccines’ (TIbVs). TIbVs would be conceived to confer a broad protection far beyond the antigens they contain. By proper targeting of innate immune cells to promote trained immunity, a TIbV may confer non-specific resistance to unrelated pathogens while trained immunity memory is still present, in addition to the specific response given by intrinsic antigens [39].
A bona fide TIbV would consist of two main components: the trained immunity inducer(s) and the specific antigen(s). The antigen(s) mission is to generate an adaptive (specific) immune response as any conventional vaccine. The trained immunity inducers aim to promote the training of innate immune cells. This innate immune training would confer non-specific resistance against unrelated pathogens for a window of time (months) plus an enhanced adaptive immune response to the antigens present in the vaccine itself or from other sources (e.g., coming from eventual infections or bystander pathogens) [39].
Two additional concepts arise under the TIbV umbrella: i) trained immunity-based immunostimulants (TIbIs) and ii) trained-immunity-based adjuvants (TIbAs). The former (TIbIs) would induce the training of innate immune cells, so they would be ready-to-act against upcoming infections conferring broad non-specific protection while trained immunity is present, still enhancing adaptive immune responses following any eventual natural infection. The latter (TIbAs) would enhance adaptive responses against specific antigens incorporated either to the trained inducers as in bona fide TIbVs, or in a separated but combined vaccine [39] (Figure 2).
Different possibilities of trained immunity-based vaccines (TIbVs). Under the umbrella of trained immunity-based vaccines (TIbVs) different possibilities exist depending on their design and purpose. Bona fide TIbVs are those containing both trained immunity inducers and antigens in the same vaccine as occurs in conventional vaccines with trained immunity inducing properties. These vaccines show heterologous protection in addition to the specific response to the target antigen. TIbIs are intended just to confer non-specific protection by means of trained immunity induction beyond the intrinsic antigens they may contain. TIbAs are intended to enhance the specific response of other vaccines that are administered later, once trained immunity has been induced, or specific antigens combined in the same vaccine as any other adjuvant.
Following the above features, the TIbV concept can be applied to existing anti-infectious vaccines composed of microorganisms that show heterologous protection ascribed to trained immunity.
During the last decades, robust epidemiological data has demonstrated the role of certain vaccines leading to protection against heterologous infection with a high impact on overall mortality in children [111, 112, 113]. This protection could not only be explained by protection achieved by the target disease. Studies on MMR vaccination in high-income settings have also evidenced a reduction in non-target infections, particularly in respiratory infections [114]. A limitation for most of these epidemiological studies is that they do not identify the agent (viral, bacterium or parasite) responsible for the infection. These heterologous effects of certain vaccines conferring non-specific protection for a quite long time are believed to be largely due to non-specific stimulation of the innate immune system. It is not yet clear whether this is a direct reflection of trained immunity induction (i.e., acting as TIbVs) in every case. The fact that most of these vaccines use live-attenuated microorganisms, i.e., self-replicating agents, may suggest that a continuous stimulation of innate immune cells is necessary to obtain protection and/or to achieve a proper trained immunity for this purpose.
The BCG-Denmark strain was tested in randomized-controlled trials (RCT) in infants who normally did not receive the BCG vaccine at birth. These studies carried out in Guinea-Bissau demonstrated that vaccination at birth was associated with lower neonatal mortality, especially due to neonatal sepsis, respiratory infections, and fever [111, 115]. In these lines, a meta-analysis commissioned by the WHO concluded that BCG administration during the first month of life reduces all-cause mortality by 30% [116]. In these studies authors did not discriminate the etiology of infection (bacterial vs. virus); therefore, a reduction in viral infections may explain, to some extent, this result. However, in two studied carried out in India in neonates with BCG-Russian strain no such effect was observed [117]; suggesting that different immunological effect of diverse BCG strains may account for these discrepancies. A study carried out in infants to assess the impact of BCG vaccination on the incidence of RSV infection suggested a possible protective role for BCG vaccination against acute lower respiratory tract infection [118]. Other clinical studies have provided evidence suggesting a protective role for BCG on secondary viral infections [79]. In this regard, the impact of BCG vaccination on viral infection in human healthy volunteers has been assessed using the live attenuated yellow fever vaccine (YFV) as a model of viral human infection [76]. BCG vaccination induced epigenetic reprogramming in human monocytes, and these modifications correlated with IL-1β upregulation and the reduction of viremia, all these features being the hallmarks of trained immunity [76].
Similar protective effect of BCG was observed in several studies in elderly people regarding respiratory tract infections. BCG vaccination in subjects of 60–75 years old once a month for three consecutive months resulted in reduction of acute upper respiratory tract infection, concomitant to significant increase in IFN-γ and IL-10 compared with those receiving placebo [119]. A recent randomized trial of BCG vaccination was carried out in elderly patients (age ≥ 65 years) returning home from hospital admission, these subjects are at high risk to develop infections. The BCG vaccination increased the time to first infection (primary outcome) and decreased the incidence of a new infection [120]. Besides, results demonstrated that BCG vaccination resulted in lower number of infections of all causes, especially respiratory tract infections of probable viral origin, although no discrimination was made between respiratory tract infections caused by bacteria or viruses.
BCG has also been shown to enhance the response to vaccines directed against viral infections [79]. A clinical study in healthy volunteers demonstrated that BCG administration prior to influenza vaccination increases antibody titers against the 2009 pandemic influenza A (H1N1) vaccine strain, concomitantly with an enhanced IFN-γ production to influenza antigens compared with the control group [121].
The cold-adapted, live attenuated influenza vaccine (CAIV) has been shown to provide non-specific cross-protection against RSV in an experimental model of infection [122].
In a randomized pilot study conducted in healthy volunteers receiving a trivalent influenza vaccine, cytokine responses against unrelated pathogens were observed [121]. During the 2003–2004 influenza A (H3N2) outbreak, an open-labeled, nonrandomized vaccine trial was carried out in children 5 to 18 years old. Subjects received either trivalent live attenuated or inactivated influenza vaccine. Live attenuated influenza vaccine but not trivalent inactivated vaccine was effective in children administered during influenza outbreak, despite the dominant circulating influenza virus was antigenically different from the vaccine strain [123].
Measles vaccine (MV) is among the live vaccines that have been shown to have beneficial effects reducing all-cause mortality [124]. Randomized clinical trials and observational studies from low-income countries have concluded that measles vaccination is associated with decreased overall mortality and morbidity [100]. However, a systematic review carried out by Higgins and colleagues has pointed out that most of these studies were considered at high risk of bias [116]. Nevertheless, MV seems to induce a transient suppressive effect on both the lymphoproliferative and innate response evaluated in peripheral blood mononuclear cells (PBMCs) from children, with slight increase in innate immune response, measured by non-specific cytokine production [100]. It has been reported that following measles vaccination, the ex vivo production of both innate (IL-6 and TNF-α) and adaptive (IFN-γ and IL-2) cytokines decreases for 2 weeks, but levels of IL-2, IL-6 and IFN-γ are increased at day 30 post vaccination compared with baseline [125]. Differences in males and females have been reported, where girls seem to receive stronger beneficial effects. In this regard, a study of MV-specific innate responses following MMR vaccination found higher TNFα, IL-6 and IFN-α secretion, cytokines associated to trained immunity, in adolescent girls than boys [126].
There are currently only three countries where polio remains endemic. Thus, polio-free, high income countries are introducing the use of the inactivated polio vaccine (IPV). However, there are still many countries that use the live-attenuated oral polio vaccine (OPV). Despite current WHO policy to replace OPV by IPV, there is epidemiological evidence that supports that replacing OPV by IPV might have an impact on overall mortality [96], since OPV has shown strong non-specific beneficial effects even in settings where the incidence of the targeted infection is low. In this regard, campaigns to eliminate polio in West Africa have been associated with lower child mortality rates [127].
As pointed above, most of the vaccines described so far showing non-specific heterologous effects contain live-attenuated microorganisms. Nevertheless, fully inactivated bacterial vaccines have also been described conferring protection against viral infections, and some of them for a fairly long period of time. Interestingly, these vaccines are mucosal preparations that are administered daily for long periods of time (weeks/months) rather than single, or seldom, doses used in live attenuated vaccines. Thus, it seems that the much longer administration of these inactivated mucosal vaccines resembles the effect achieved by live vaccines on heterologous protection associated to trained immunity (Figure 3).
Trained immunity window by self-replicating and inactivated TIbVs. Trained immunity-based vaccines (TIbVs) containing live-attenuated self-replicating microorganisms (e.g. BCG) may require fewer administrations to induce an adequate trained immunity window of sufficient intensity, quality and/or duration than vaccines with dead microorganisms. Fully-inactivated TIbVs can be enhanced to induce trained immunity with a multiple dose schedule (e.g. MV130).
These vaccines are used for the prevention of recurrent infections in susceptible subjects, mainly associated to the respiratory and urogenital tracts [128, 129, 130, 131, 132, 133, 134]. Since they target infections occurring in these tracts, their administration is generally through mucosal tissues to obtain a better mucosal response [135, 136].
MV130 is a sublingual vaccine used to prevent recurrent respiratory tract infections [128, 129] containing inactivated whole-cell bacteria that are common pathogens in the airways. Its ability immunomodulating DCs has been addressed experimentally in vitro and in vivo. MV130 triggers the release of cytokines ascribed to trained immunity in different setting, including TNF-α, IL-1β and IL-6 [103, 137, 138]. Sublingual immunization of mice with MV130 induces a systemic Th1/Th17 and IL-10 enhanced responses against unrelated antigens [103]. Similar enhancement was shown in patients treated with MV130 where an increased T cell response to flu antigens were described [128]. MV130 was successfully used in infants with recurrent wheezing, a condition triggered in most cases by viral infections. It is noteworthy that the protective effect was also shown 6 months after discontinuation of treatment, which points to a long-lasting effect that fits with the memory ascribed to trained immunity (Nieto et al., under review). In this regard, MV130 has been shown to induce trained immunity and to confer protection against experimental virus infections (Brandi et al., under review). Recent studies have assessed the clinical benefit of MV130 as a TIbV in the context of recurrent respiratory infections in vulnerable populations such as patients with different primary and secondary immunodeficiencies showing a reduced rate of respiratory infections [130, 139] (Ochoa-Grullón et al., in press).
Although not considered vaccines but immunostimulants, these bacterial preparations are, like MV130, used for the prevention of recurrent respiratory infections. OM-85, one of the best studied, is composed of chemically treated bacterial lysates for oral administration, acting through the gastro-intestinal mucosa. OM-85 has been shown to be effective in experimental viral infections [140] and in children with recurrent wheezing [141], a condition triggered by viruses as noted above. OM-85 stimulates the release of proinflammatory cytokines such as IL-1β, TNF-α and IL-6 by macrophages [142], typical of trained immunity induction, as well as Th1 cytokines including IFN-γ [143]. It is not known, however, the role of trained immunity in their mechanism of protection. A recent study conducted in infants, the observed protection against respiratory infections under OM-85 treatment stopped when treatment was discontinued [144], which may point against the memory ascribed to trained immunity.
The non-specific mechanism of TIbVs against widely differing pathogens associated to the induction of trained immunity can be exploited clinically. This makes TIbVs as a ready-to-act tool to tackle disease outbreaks from different angles where conventional specific vaccines have proven their limitations:
Newly emerging disease outbreaks, with no conventional vaccines available. Even in the presence of therapeutic options, vaccines are the best tool to prevent infections. However, even with worldwide efforts, getting a vaccine to the public takes time. In addition, side effects, dosing issues, and manufacturing problems can all cause delays [3]. Herein, using available TIbVs could mitigate the devastating consequences of emergent outbreaks by means of non-specific protection, until a suitable specific vaccine is available.
Newly emerging disease outbreaks, first coming vaccines with partial efficacy. Even if a vaccine gets available to the market, conventional strategies might raise some issues. The unpredictable identity of largely unknown emerging pathogens, the lack of appropriate experimental animal models, and the time for faster developing may give raise to an upcoming vaccine with no full efficacy [3]. On the other hand, limitations of current vaccines, such as mumps, also include a low efficacy resulting from an unacceptable drop in the immune response over time, requiring re-immunization [145]. In these contexts, the administration of a TIbV prior to the specific vaccine may enhance the response to the latter (111).
Re-emerging disease outbreaks, pathogens with high mutation rates and loss of vaccine efficacy. Mutations are the building blocks of evolution in any organism. Viruses are among the fastest evolving entities, especially RNA viruses such as influenza. Implications in conventional vaccine design are numerous, as a high mutation rate makes it hard to design a vaccine that is universally effective across many years. As a result, this makes a vaccine effective for shorter and raises the need for yearly vaccination programs [22, 146]. Since the underlying mechanism of TIbVs extend well beyond their nominal antigens and have a broad-spectrum of protection, TIbVs could overcome the troublesome of highly specific vaccines that promote antigen variant switching [147].
Disease outbreaks in vulnerable populations. During infectious disease outbreaks, vulnerable populations are usually disproportionately affected due to an interplay of immunological, epidemiological, and medical factors, which leads to sub-optimal or even under-vaccination [148]. This is well exemplified in the elderly population, where successful vaccination against important infectious pathogens which cause high morbidity and mortality represents a growing public health priority. Age-related immunosenescence and ‘inflammaging’ have been postulated as underlying mechanisms responsible for decreased response and high mortality, including during COVID-19 pandemic or influenza season [80, 149]. Therefore, more potent vaccines are needed. In this regard, the induction of trained immunity by the use of TIbVs is proposed to overcome the immune dysfunction found in these individuals [28]. Thus, elderly has been proposed as one of the groups to benefit from the use of TIbVs, including severe COVID-19 disease, with the aim of potentiating the immunogenicity of their vaccination [80]. Moreover, some types of immunodeficiencies or immunosuppression may benefit from TIbVs. These formulations, by means of tackling both branches of immunity, especially the innate compartment, may be an achievable alternative to reinforce protection or optimize immunogenicity of vaccination in this population [130, 139].
Altogether, harnessing the TIbV concept has been suggested as a crucial step in future vaccine development and implementation, because a wide range of clinical applications may benefit to some extent from their use [150].
Despite the tremendous financial and scientific effort invested to rapidly obtain a prophylactic vaccine against SARS-CoV-2, only the first one has been licensed in December 2020. Although this means less than a year since the declaration of the pandemic by the WHO, which is an unprecedented achievement, in the meantime, two pandemic waves of COVID-19 and more than 1.5 million deaths have been declared worldwide. Therefore, alternative strategies have been considered to fill the gap until a safe and effective vaccine is available. As noted earlier in this chapter, TIbVs can play an important role for this purpose by increasing host resistance to other pathogens, including viruses.
A bunch of recent studies have been published supporting the role of certain vaccines, including BCG, OPV and measles, as a possible successful strategy to reduce susceptibility and severity to SARS-CoV-2 through trained immunity induction [80, 151, 152]. Thus, clinical trials are currently being conducted to find out the contribution of trained immunity as a preventive tool in the context of COVID-19 pandemics [153]. In a prospective observational trial, 255 MMR vaccinated subjects were followed searching for COVID-19 cases, thirty-six presented COVID-19 but all with a remarkable mild course [154]. Recent studies have also suggested a potential benefit of influenza vaccine on the susceptibility and the outcome of certain infections including SARS-CoV-2. In this sense, a particular attention has been focused on a high-risk population, the elderly. In a study conducted in Italy, influenza vaccination in people aged 65 and over was associated with a reduced spread and a less severe clinical expression of COVID-19 [155].
Finally, in addition to the potential role of TIbV conferring resistance against SARS-CoV-2 infection, they can eventually be used to increase efficacy of specific anti-COVID-19 vaccines, when available, especially in vulnerable population. In this sense, implications of vaccination route and mucosal immunity have also been raised as a key aspect in the development of safe and effective prophylaxis interventions against SARS-CoV-2. Most formulations in development are parentally administered; only a few COVID-19 vaccine candidates are administered by mucosal routes. Still, studies indicate that even if mucosal immunization against coronavirus does not confer sterilizing immunity, the ability to induce anti-SARS-CoV-2 IgA responses in the airways may prevent virus spread to the lung and avoid respiratory distress [156]. In this regard, mucosal TIbVs could enhance the mucosal response of specific COVID-19 vaccines acting as TIbAs by combining them as pointed above in those especially vulnerable subjects.
Viral outbreaks can cause epidemics and pandemics if the route of transmission allows for the rapid virus spread. Given the ease of travel and the global exchange of potential transmitting agents, these situations will be increasingly frequent in the future. Preventing the spread of a virus outbreak caused by a highly contagious agent is not easy in the absence of effective therapies or preventive measures. Although the development of effective prophylactic vaccines specific for the threatening virus is the final goal when possible, this requires a minimum time of almost a year in the best possible scenario. Meanwhile, the consequences of the spread of a deadly virus can be devastating, as it is exemplified during the COVID-19 pandemic. This scenario may also take place in the case of re-emerging viruses tackled by partial efficacious vaccines. In such situations, harnessing the heterologous non-specific protection of some existing vaccines with a known safety track record is an interesting possibility. This protection may be critical for vulnerable subjects and/or for highly exposed individuals, like healthcare workers.
Non-specific protection of some vaccines is thought to be mainly dependent on their effect on the innate immune system. Increasing evidence gathered over the past few years points that innate immune cells show memory-like features when properly activated. This memory termed “trained immunity” has been associated with the non-specific protection of vaccines. The concept of “trained immunity-based vaccine” (TIbV) has been drawn to exploit the potential of trained immunity in designing novel vaccines or to redefine bacterial-derived preparations conferring broad protection against widely differing pathogens. As trained immunity may have implications on the adaptive immune response and vice-versa, its potential to provide enhanced immune responses is quite broad whether considering natural infections or following vaccination.
Taken advantage of the current COVID-19 pandemic, a number of clinical trials have been launched with putative TIbVs in order to address protection in highly exposed subjects. The results are eagerly expected as these initiatives may be considered as a proof-of-concept supporting their use in future epidemics/pandemics to fill the gap until a specific vaccine is available. Nevertheless, as trained immunity can be achieved by different inducers, it is unlikely to obtain the same degree of protection, duration, etc. for all of them, which may also depend on the biological behavior and the route of transmission of the threatening pathogen. As in most instances rapidly spreading viruses are airborne and primarily infect the mucosa of the airway tract, induction of trained immunity at the local mucosal level can confer a more adequate protection. This may be an opportunity for mucosal TIbVs as compared to those given parenterally.
Trained immunity may justify heterologous protection of vaccines, help to explain their underlying mechanisms, open avenues for next generation of vaccines, or be proposed to tackle outbreaks by new pathogens as described here. However, this is an emerging field that requires more clinical data before being a reality in the clinical practice; not only to be used against infectious outbreaks, but to fight against recurrent infections in vulnerable subjects for whom no effective vaccines are yet available.
JLS is the founder and CEO of Inmunotek SL, Spain, a pharmaceutical company that manufactures bacterial vaccines. LC and PS-L are employees of Inmunotek.
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