# Heat inactivated mycobacteria, alpha-Gal and zebrafish: Insights gained from experiences with two promising trained immunity inductors and a validated animal model

Ramón Juste, Elisa Ferreras-Colino, José de la Fuente, Mercedes Domínguez, María Risalde, Lucas Domínguez, Alejandro Cabezas Cruz, Christian Gortázar

<figure><img src="image_1.png" /></figure>

To cite this version:

Ramón Juste, Elisa Ferreras-Colino, José de la Fuente, Mercedes Domínguez, María Risalde, et al. Heat inactivated mycobacteria, alpha-Gal and zebrafish: Insights gained from experiences with two promising trained immunity inductors and a validated animal model. Immunology, 2022, 167 (2), pp.139-153. 10.1111/imm.13529. hal-03993747

HAL Id: hal-03993747

https://hal.inrae.fr/hal-03993747

Submitted on 17 Feb 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
REVIEW

<figure><img src="image_3.png" /></figure>

# Heat inactivated mycobacteria, alpha-Gal and zebrafish: Insights gained from experiences with two promising trained immunity inductors and a validated animal model

Ramón A. Juste<fnref n="1" />,<fnref n="2" />

Elisa Ferreras-Colino<fnref n="3" />

<figure><img src="image_4.png" /></figure>

José de la Fuente<fnref n="3" />,<fnref n="4" />

Mercedes Domínguez<fnref n="5" />

María A. Risalde<fnref n="6" />,<fnref n="7" />

Lucas Domínguez<fnref n="8" />,<fnref n="9" />

Alejandro Cabezas-Cruz<fnref n="10" />

Christian Gortázar<fnref n="3" />

**Correspondence**

Elisa Ferreras-Colino, SaBio, Instituto de Investigación en Recursos Cinegéticos IREC-CSIC-UCLM-JCCM, Ronda de Toledo 12, 13005 Ciudad Real, Spain.
Email: elisa.ferreras@uclm.es

**Funding information**

European Regional Development Fund, Grant/Award Number: BIOGAL
PID2020-116761GB-I00; Gobierno del Principado de Asturias, Grant/Award
Number: PCTI 2021-2023 (GRUPIN: IDI2021-000102; Junta de Comunidades de Castilla-La Mancha, Grant/Award
Numbers: CCM17-PIC-036
(SBPLY/17/180501/000185),

**Abstract**

Trained immunity (TRAIM) may be defined as a form of memory where innate immune cells such as monocytes, macrophages, dendritic and natural killer (NK) cells undergo an epigenetic reprogramming that enhances their primary defensive capabilities. Cross-pathogen protective TRAIM can be triggered in different hosts by exposure to live microbes or microbe-derived products such as heat-inactivated *Mycobacterium bovis* or with the glycan α-Gal to elicit protective responses against several pathogens. We review the TRAIM paradigm using two models representing distinct scales of immune sensitization: the whole bacterial cell and one of its building blocks, the polysaccharides or glycans. Observations point out to macrophage lytic capabilities and cytokine regulation as two key components in non-specific innate immune responses against infections. The study of the TRAIM response deserves attention to better characterize the

**Abbreviations:** AGS, alpha-Gal syndrome; AKR2, akirin-2; BCG, Bacille Bilié Calmette-Guerin; COVID-19, coronavirus disease 2019; GalTG, galactosyltransferase; H3, histone 3; HIMB, heat-inactivated *Mycobacterium bovis*; IL-6, interleukin-6; LncRNA, long non-coding RNA; LPS, lipopolysaccharides; miRNA, microRNA; NFkN, nuclear factor kappa-light-chain-enhancer; NK, natural killer; NLR, nucleotide-binding oligomerization domain-like receptor; PAMP, pathogen-associated molecular pattern; PRR, pattern recognition receptor; SARS-CoV-2, Severe acute respiratory syndrome coronavirus 2; SUB, subolesin; TLR, toll-like receptor; TNF, tumour necrosis factor; TRAIM, trained immunity; α-Gal, Galα1-3Galβ1-(3)4GlcNAc-R.

This is an open access article under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2022 The Authors. *Immunology* published by John Wiley & Sons Ltd.

<footnote n="1">¹Animal Health Department, NEIKER-Instituto Vasco de Investigación y Desarrollo Agrario, Derio, Bizkaia, Spain</footnote>

<footnote n="2">²NySA, Servicio Regional de Investigación y Desarrollo Agroalimentario, Villaviciosa, Asturias, Spain</footnote>

<footnote n="3">³SaBio, Instituto de Investigación en Recursos Cinegéticos IREC-CSIC-UCLM-JCCM, Ciudad Real, Spain</footnote>

<footnote n="4">⁴Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, Oklahoma, USA</footnote>

<footnote n="5">⁵Unidad de Inmunología Microbiana, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain</footnote>

<footnote n="6">⁶Departamento de Anatomía y Anatomía Patológica Comparadas y Toxicología, Facultad de Veterinaria, Universidad de Córdoba, Córdoba, Spain</footnote>

<footnote n="7">⁷CIBERINFEC, ISCIII – CIBER de Enfermedades Infecciosas, Instituto de Salud Carlos III, Madrid, Spain</footnote>

<footnote n="8">⁸VISAVET Health Surveillance Centre, Complutense University of Madrid, Madrid, Spain</footnote>

<footnote n="9">⁹Department of Animal Health, Faculty of Veterinary Medicine, Complutense University of Madrid, Madrid, Spain</footnote>

<footnote n="10">¹⁰UMR BIPAR, INRAE, ANSES, Ecole Nationale Vétérinaire d'Alfort, Université Paris-Est, Maisons-Alfort, France</footnote>
<figure><img src="image_5.png" /></figure>

MYCOTRAINING

SBPLY/19/180501/000174; Ministerio de
Ciencia e Innovación, Grant/Award

Number: MCIN/

AEI/10.13039/501100011033; Universidad
de Castilla-La Mancha, Grant/Award

Number: 2020/3836

evolution of host-pathogen cooperation both for identifying the aetiology of some
diseases and for finding new therapeutic strategies. In this field, the zebrafish pro-
vides a convenient and complete biological system that could help to deepen in
the knowledge of TRAIM-mediated mechanisms in pathogen-host interactions.

KEYWORDS

cross-protection, glycan alpha-Gal, heat-inactivated *Mycobacterium bovis*, macrophages, trained innate immunity

INTRODUCTION

The development of the classical models of specific
immunity revolutionized the field of medicine [1, 2].
They allowed not just to diagnose infectious diseases
quickly and specifically, up to then the main causes of
human and animal mortality, disability and suffering in
general, but contributed to prevent and cure them. These
models reversed the impact of evolutionary pressure for
resilience brought up by the large and dense social urban
way of life and allowed an unprecedented increase in
human population. The cumulated knowledge in the
field of infectious diseases and immune responses has
meanwhile defined a grey area of phenomena that do not
fit the specific memory paradigm of adaptive immunity
because of involving non-specific immune memory.
Trained immunity (TRAIM) is a newly proposed model
of immune response that has come to fill in the gap and
to explain those immune phenomena that do not fit the
classical features of the powerful adaptive immune
defence: specificity, memory, clonal selection mechanism
and presence only among the vertebrates in the evolu-
tionary tree [3]. Its insights can lead to a better assess-
ment of some inflammatory phenomena as well as to
develop better vaccines by understanding the reasons of
failure and protection as well as the role of adjuvants.

In this review, we focused on the analysis of the
TRAIM paradigm using two models we use in our
research and representing two distinct scales of immune
sensitization: the whole bacterial cell and one of its build-
ing blocks, the polysaccharides or glycans. While review-
ing the subject can help us to better understand our own
experience and to overcome the observed problems, we
think we can also contribute to the buildup and support
of an idea that can have important implications in
medicine.

TRAINED IMMUNITY

TRAIM may be defined as a form of memory where
innate immune cells such as monocytes, macrophages,
dendritic and natural killer (NK) cells undergo an

epigenetic reprogramming that enhances their primary
defensive capabilities [3–5] (Figure 1). This reprogram-
ming or modification of gene expression control affects
immune-related mitochondrial and metabolic functions
and is a consequence of exposure to a primary pathogen
stimulus. When a homologous or heterologous-related sec-
ondary stimulus happens again, the result is an enhanced
immune response [6–10]. It has been shown that exposure
to live agents such as bacille Bilié Calmette-Guerin (BCG)
or microbe-derived products such as heat-inactivated
extracts, lipopolysaccharides (LPS), chitosans or β-glucans
can reprogram or train the innate immune system for
TRAIM-mediated responses to secondary stimuli [7, 11].
The difference between this type of memory and the con-
ventional adaptive one is that, instead of lymphocyte clonal
selection, this type of immune memory depends on genetic
modification of the cells involved. This takes place through
histone 3 (H3) modifications and microRNA (miRNA)
release associated with chromatin remodelling and epige-
netic reprogramming [6, 12]. A relevant difference in adap-
tive versus TRAIM response routes is that the former is
conformed against exquisitely elaborate singular tridimen-
sional amino-acidic structures, while the latter is driven by
larger highly repetitive widespread microbe-associated pat-
terns [3].

Despite the old empiric knowledge that suffering the
infection with a certain pathogen can help to fight
against others, the first experimental evidence of what
today is known as TRAIM dates back to an ex-vivo assay
carried out in 1957 by Elberg et al. [13], in which it was
shown that contact with *Mycobacterium tuberculosis*
(*M. tuberculosis*) induced a higher cell survival to subse-
quent in vitro challenge with bacteria of the genus
*Brucella*. The concept of this cross-protection was further
demonstrated a few years later by Mackaness with
*Brucella* and *Listeria* [14] and then reviewed in a broader
perspective by Allison [15]. This provided a mechanistic
basis for the earlier observed non-specific effects of BCG
vaccination on child mortality, but was not studied until
recently related to this and to other vaccines [16–19].

This enhanced immune resistance seems to have a
genetic component related in part to adaptive immune
memory due to exposure to different pathogens by
<figure><img src="image_6.png" /><figcaption>**FIGURE 1** Proposed mechanisms of TRAIM. Upon effective contact of a pathogen-associated molecular pattern (PAMP) with the myeloid cell pattern recognition receptors (PRRs), epigenetic reprogramming (through long non-coding RNA: LncRNA) is induced by modifications in the histone monomer H3 that loosens the DNA packaging making it more accessible for transcription or not depending on the type of stimulus (tolerance [LPS] or training β-glucan or BCG]). As a result, macrophages encountering the same patterns, no matter on which specific agent, are able to mount a response that might be less efficient if the priming was of a tolerizing type or more efficient if the priming was of the training type. Naïve individuals will respond at the basic level of efficiency. TRAIM, trained immunity.</figcaption></figure>

crowded life in sedentary populations [20] and their
accompanying domestic animals. This response is likely to
be caused by a primitive mechanism that is present in the
whole evolutionary scale, but that will better compare with
social insects and that was recently recognized to be an
important player also in vertebrates [3]. Social insects like
ants and bees are under heavy infectious pressure given
their clonic genetic population composition and crowded
type of life. In addition to mechanic and behavioural
defence activities, epigenetic reprogramming is a widely
used mechanism for social insect polyphenism [21] that has

been applied to defence against microorganisms allowing
transmission of strengthened responses against pathogen
recognition patterns [22–25]. Such successful mechanisms
of improving immune fitness would have been conserved
along the evolutionary chain, but not explicitly recognized
nor studied until the recent enunciation of the TRAIM con-
cept [3]. Although there are different cells capable of speed-
ing up offending agent elimination as it occurs in
invertebrates and plants [26], the hallmark of active innate
immune responses in vertebrates is an efficient lytic activity
and phagocytosis by macrophages [3, 27–29].
<figure><img src="image_7.png" /></figure>

Epigenetic reprogramming and its
metabolism

Gene expression is regulated by promoters (proximal
transcription elements) and enhancers (distal transcrip-
tion elements). The action of these elements can be regu-
lated without DNA sequence modification through three
main processes that have been shown to be used by
TRAIM: DNA methylation [30, 31], histone proteins con-
figuration [32] and non-coding RNAs [6, 33]. Balance
between these mechanisms and reversion of their
changes are what determine the degree of enhancement
of the response and its duration in time. For instance, it
has been shown that trimethylation of lysine 4 at histone
3 marks active promoters while its monomethylation
mark enhancers [34]. These marks, as well as the DNA
methylation changes lead to the unfolding of the chroma-
tin thus speeding up transcription and expression of pro-
inflammatory factors. Since these changes are only par-
tially reversed when the triggering cause disappears, the
secondary challenge initiates a quicker and stronger gene
expression. How these mechanisms are used in the con-
text of TRAIM has been studied in a series of studies by
the Radboud Institute for Molecular Life Sciences [12,
32, 35], who also demonstrated the involvement of two
specific enzymes in β-glucan training, the KDM5 family
of histone demethylases that is inhibited and thus favours
persistance of training [36] and Set7 that writes a histone
3 lysine 4 monomethylation [37] that accelerate the Tri-
carboxilic acids cycle and the oxidative phosphorylation
pathways that are used to fuel the trained immune
responses.

Phagocytosis

Phagocytosis as a relevant process in tissue defence
against infections was defined by Metchnikoff [38].
Phagocytosis can be found in unicellular organisms
where it is part of the normal cell metabolism, in inverte-
brates with specialized and non-specialized cells, and also
in vertebrates where it is essentially carried out by cells
of the myeloid lineage that mature into specialized
mature macrophages whose evolution can further pro-
gress to dendritic, epithelioid and multinucleated cells
[39]. No specific stimuli seem to be needed to trigger
phagocytosis, but complex mechanisms are responsible
for the degradation of the pathogens in the lysosomes
[40]. This gives the macrophages a plasticity that is a key
factor in both innate and adaptive immune defence and
can be activated by two main routes: the classical one,
stimulated by microbial toll-like receptors (TLRs) ligands
and interferon-gamma (IFNγ), and the alternative one,

triggered by interleukin (IL)-4/IL-13. These routes cause
a polarization of the macrophage functionality into differ-
entiated phenotypes defined as M1 and M2, respectively
[41–43]. The M1 is characterized by the release of pro-
inflammatory cytokines and high amounts of reactive
oxygen and nitrogen intermediates associated with the
promotion of a Th1 response with strong anti-microbial
and inflammatory activity [42, 44, 45]. The M2, on the
contrary, shows an anti-inflammatory profile, tissue
remodelling, tumour growth and immunoregulatory
functions [28, 38, 46]. From these cellular mechanisms, a
wide range of positive consequences, such as pathogen
clearing and tissue repair in successful mode, and nega-
tive consequences such as pathogen persistence and
chronic inflammation in tolerizing mode, emerge
depending on external (type of pathogen or intensity of
exposure) and internal (genetics or activation route) fac-
tors [47]. The former consequences are those associated
with the M1 phenotype and constitute the genuine goals
of TRAIM. The latter, linked to the M2 phenotype, would
be the collateral damages driven by factors like insulin
and obesity that confer TRAIM mechanisms a central
role not only in infectious agents defence, but also in
some of the current most common chronic inflammatory
diseases of metabolic origin [34, 42].

Cytokines

Other factors relevant for innate immunity that may be
involved in TRAIM include complement component
3 (C3) and akirin-2 (AKR2) [48–50]. Complement recep-
tor 3 mediates activation of innate immune cells in
response to β-glucans (a specific type of glycans com-
posed only by monosaccharides) and triggers the macro-
phage tumour necrosis factor (TNF) and IL-6 response to
induce TRAIM [51]. Another possible mechanism of
TRAIM includes the stimulation of the immune system
through pattern-recognition TLRs [52]. In this sense,
when monocytes and NK cells from BCG-vaccinated
individuals are compared to non-vaccinated controls,
they display higher TLR and cytokine expression levels in
response to various pathogens (e.g., *Bacillus anthracis*,
*Brucella suis*, *Staphylococcus aureus*, *Pasteurella pestis*,
*Listeria monocytogenes*, *Klebsiella pneumonia*, etc.) and
their products (β-glucans, lipoproteins, LPS, flagellin,
muramyl dipeptide, etc.) [7, 53]. Standardizing methods
to measure TRAIM is urgently needed to continue
research in this area. In this sense, simple M1/M2 polari-
zation balance macrophage characterization could be a
first approach that, further developed with ex-vivo phago-
cytosis assays, would lead to simplified TRAIM marker
assays. Three interesting candidates recently appearing to
<figure><img src="image_8.png" /></figure>

have a potential in a killed mycobacterial model could be iNOS, IL10 and MIP-1β [54] that, in an ex-vivo macrophage paratuberculosis vaccination goat model, showed significant increases (iNOS and IL10) or decreases (MIP-1β) relative to controls, but no differences after secondary ex-vivo macrophage challenge with *M. avium* subsp. *paratuberculosis*.

## TRAIM INDUCTORS

As pointed out above, TRAIM is a primitive mechanism of eukaryotes that has been maintained throughout the whole evolutionary tree up to its highest branches. It probably appeared as an advantage to respond to the loss of fitness caused by other organisms evolving to parasitism and trying to steal oneself resources. Therefore, it had to adapt pre-existing metabolic mechanisms to recognition of general patterns of those varied range parasitic organisms. Therefore, primitive organism had to be able in the first place to respond to the challenge by increasing energy and materials production at the cellular level through glycolysis oxidative phosphorylation, tricarboxylic acids cycle, pentose phosphate pathway, fatty acid oxidation, fatty acid synthesis and aminoacid metabolism in the fight to destroy the invading parasite. If successful, the next step to keep the gained evolutionary advantage would be to maintain such readiness until the next encounter with the parasite or even, if possible, to any other with similar structural characteristics. This would lead to a sharing of similar response mechanisms after exposure to molecular triggers present in different biological agents. Bacterial and fungal cells and their components (LPS, β-glucan and chitin), virus and parasites are the best-known exogenous ones, but there are other endogenous ones like oxidized low-density lipoprotein, apolipoprotein(a), aldosterone or adrenaline [34]. Since training or tolerizing effects are observed, it must be underscored that TRAIM is a complex delicately balanced mechanism that is only beginning to be understood, and that the same compound, depending on time and concentration might cause opposite effects [53, 55]. In this review, we will focus on two examples: on one side, a whole bacterial cell, *Mycobacterium bovis* inactivated vaccine which would be the closest approach to a natural challenge without its risks, and on the other, one structural component of those types of organism found in bacterial cell wall a broadly known as glycan, an encompassing term grouping large glycosidically bound saccharide polymers including those linked to lipids or proteins, that probably represents the type of true molecular effector of TRAIM present in the former in more complex forms.

## Mycobacteria

The BCG vaccine, an attenuated *M. bovis* strain, was first introduced in humans in 1921 and is still the only registered vaccine to prevent tuberculosis (TB) [56]. Both epidemiological and experimental studies concerning BCG suggested the first glimpses of TRAIM. Beyond its specific protective effect against disseminated forms of TB in infants, extensive to leprosy [57, 58], attention has been recently drawn to early and reiterated epidemiological studies that show a decrease of overall mortality rate in BCG-vaccinated children that is larger than that attributable to TB itself [59]. This reduction in infant mortality is mainly associated to BCG-induced cross-protection against unrelated pathogens, especially sepsis and respiratory infections [60, 61]. Furthermore, BCG also provided the first experimental evidence of TRAIM [62], and has become one of the most studied TRAIM inducers [18, 53, 63–71]. Over the last decades, several experimental studies have reported that BCG stimulation induces protection upon a secondary encounter with unrelated pathogens such as herpes simplex virus [72, 73], influenza virus [72, 74, 75], *Staphilococcus aureus* (*S. aureus*) [64, 76], *Salmonella enteritidis* [62], *Leishmania major* [77], *Plasmodium* spp. [78], *Trypanosoma cruzi* [79], *Babesia microti* [80] and *Candida albicans* (*C. albicans*) [64, 81] in murine models, as well as yellow fever virus [82], human papillomavirus [83] and *Plasmodium* spp. [84] in humans. Although adaptive immunity is also likely to participate [85], the speed at which responses appear (few days to 1 week after vaccination), and the particularities of the infant immune system (e.g., adaptive immunity not being fully mature), strongly support the hypothesis of the innate immune system playing a major role in the observed non-specific effects following BCG vaccination [60]. In this respect, after immunization with BCG and challenge with unrelated pathogens, circulating monocytes/macrophages and NK cells display an increased capacity of secreting pro-inflammatory cytokines in a lymphocyte-independent manner [64, 81]. Two molecular mechanisms seem to be involved in the induction of a trained phenotype in innate cells after stimulation with BCG. First, epigenetic reprogramming through histone modifications, concretely TRAIM methylation of lysine 4 in histone 3 (H3K4me3), which is associated with gene transcription [86], occurs at the promoters of genes encoding immunological markers [81]. These epigenetic modifications are dependent on the NOD2 receptor, which is present in monocytes [81]. Second, cell metabolism shifts from oxidative phosphorylation to aerobic glycolysis, a.k.a. Warburg effect, in trained monocytes/macrophages and NK cells [87]. In fact, fumarate and mevalonate, which are metabolites derived from glutaminolysis and cholesterol synthesis,
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**FIGURE 2** Induction by HIMB immunostimulant of protective responses to multiple pathogens. HIMB significantly decreased the number of mycobacteria per granuloma and the number of granuloma, as well as increased the expression of C3 and IL-1b in zebrafish vaccinated intraperitoneally or by immersion and challenged with *Mycobacterium marinum* (*M. marinum*) [99, 104]; significantly reduced bacteria/parasite burden in mice vaccinated orally and challenged with *Borrelia burgdorferi*, *Plasmodium* sp. and *Leishmania amazonensis* in mice, and significantly increased weight and reduced clinical signs and lesions in pigs vaccinated orally and challenged with *Salmonella enterica* [105]. HIMB, heat-inactivated *M. bovis*.</figcaption></figure>

respectively, induce enrichment of H3K4 at the promoters of several cytokine-encoding genes. Thus, epigenetic regulation and metabolic pathways seem to operate jointly [18].

Despite the extensive safety record of BCG vaccination both in humans [88], domestic animals and wildlife species [89], disseminated BCG infection may occur in immunocompromised individuals [90]. In addition, the possibility of excretion into the environment after oral vaccination and the necessity of maintaining the cold chain must be considered when delivering live vaccines [91]. Therefore, immunostimulants based on inactivated mycobacteria displaying BGC-like effects would constitute a more environmentally safe and easily handled approach to induce protection against TB [92, 93], and cross-protection against unrelated pathogens in human and animal populations.

Garrido et al. [93] developed an immunostimulant based on heat-inactivated *M. bovis* (HIMB) which has demonstrated to reduce the mycobacterial load in target tissues and tuberculous lesions in cattle [94], goats [95], pigs [96], wild boar (*Sus scrofa*) [93], red deer (*Cervus elaphus*) [97] and European badger (*Meles meles*) [98]. In the cited experiments, the HIMB formulation, alone or in combination with adjuvants, was administered either via

oral or parenteral. The protective capacity of HIMB has been correlated with innate-response markers TLRs, complement factors and pro-inflammatory cytokines [91, 99]. In addition, C3 was proposed as a possible correlate of natural resistance to *M. bovis* infection in wild boar [100]. Furthermore, immunization of calves with HIMB enhanced the capacity of monocyte-derived macrophages to destroy *M. bovis* in vitro, an effect that was independent of cellular or humoral adaptive immune responses and thus coherent with TRAIM [101]. This is consistent with the observation of macrophage-mediated *M. leprae* destruction in TB-infected patients [40]. As a matter of fact, immunization with HIMB has shown protective capacity not only against *Mycobacterium* infection but also against unrelated pathogens such as *Plasmodium* and *Leishmania* in mice [102] and *Salmonella* in swine [103] (Figure 2). Although immunization with HIMB did not reduce *S. enterica* serotype Choleraesuis burden, immunized pigs showed higher levels of pro-inflammatory cytokines such as serum TNFα and lung CCL28, lower levels of lipid oxidation, and higher activity of antioxidant enzymes than non-immunized pigs [103]. In connection with this, pre-exposure to Freund's complete adjuvant containing inactivated *M. bovis* was also
found to be protective against *Pasteurella piscicida* infection in Yellowtail fish [106]. Furthermore, when formulations containing HIMB, alone and in combination with recombinant Subolesin (SUB), the tick ortholog of human AKR2; were orally administered to cattle experimentally infested with cattle ticks *Rhipicephalus microplus*, a significant reduction in the number and fertility of female ticks in cattle immunized with HIMB+SUB compared to HIMB-immunized animals was observed [107]. These findings are consistent with the known utility of *M. bovis* as an immune adjuvant. Indeed, the immunization with HIMB alone also suggested a reductive effect on female tick weight and oviposition, and the analysis of mRNA levels of immune response markers showed upregulation of both innate and adaptive immunity [107]. The innate mechanisms were mediated by TLRs through upregulation of AKR2, IL-1β, C3 and TNFα; while the adaptive response was mediated by anti-SUB antibodies, which is, in fact, the best documented protective effect of tick vaccines [108]. Overall, the abovementioned results suggest that immunization with HIMB, in addition to specific cellular and antibody-mediated adaptive immunity, can also activate innate immune mechanisms and TRAIM to induce not only protection against mycobacteria but also cross-protection against other pathogens [7, 48–50, 53, 109].

## Glycans

Contrary to the fine tridimensional singularity lending exquisite species or even lineage specificity to adaptive immune response epitopes of proteins, glycans are highly repetitive patterns shared by many cell structures throughout both pathogenic and non-pathogenic microorganisms. The extensive presence of glycans in pathogens is responsible for the lack of specificity that characterizes the innate immune responses targeting them [53]. Although the role of glycans in innate immunity has been reported, questions remain regarding their role in immune regulation and protection against pathogen infection [110]. β-Glucans in general, and fungal-derived ones, are by far the most studied glycans as innate response modulators. Several decades ago, Di Luzio et al. [111] and Bistoni et al. [112] reported reduced lethality in mice due to *S. aureus* and *C. albicans* after administration of *S. cerevisiae*- or *C. albicans*-derived β-glucans, respectively, through B/T lymphocytes-independent mechanisms [112]. Their findings were subsequently corroborated in recent experiments [113], in which the survival rate to lethal *C. albicans* infection was increased in both wild-type and T/B lymphocytes deficient mice previously infected with low-dose β-glucan.

Conversely, this effect was not observed in monocyte deficient mice, suggesting a key role of monocytes/macrophages in the protective immune mechanisms [111, 113, 114]. Furthermore, in vitro production of IL-6 and TNF-α by human peripheral blood mononuclear cells and purified monocytes were enhanced after incubation with *C. albicans* or β-glucans in a dose-dependent manner and up to 2 weeks [113]. Although the molecular mechanisms involved are not fully elucidated, epigenetic programming and metabolic shift also seem to play a crucial role in the innate system training by β-glucans [5, 113, 115].

Despite the glare of glucans among glycans, here we will review evidence of the immunological relevance of a less known molecule. The glycan Galα1-3Galβ1-(3) 4GlcNAc-R (α-Gal), present in tick salivary glycoproteins and non-catarrhine mammalian cells, has recently been associated with the alpha-Gal syndrome (AGS) that causes delayed IgE-mediated anti-α-Gal anaphylaxis to mammalian meat consumption and immediate anaphylaxis to xenotransplantation, certain drugs such as cetuximab, and tick bites [116, 117]. Humans do not produce α-Gal and natural anti-α-Gal IgM/IgG antibodies are produced in response to gut microbiota with this glycan on bacterial surface [118]. The hypothesis is that humans evolved by losing the capacity to synthesize α-Gal thereby acquiring the capacity to develop a strong antibody response against this glycan that is protective against pathogens containing this modification [119]. As humans, fish and birds do not synthesize α-Gal and display natural antibody levels to this glycan likely in response to bacterial gut microbiota [120, 121]. Accordingly, in the α-Gal-negative galactosyltransferase (GalT)-KO mouse and zebrafish (*Danio rerio*) animal models, immunization with this glycan boosts immune protective mechanisms against multiple pathogens [122]. In experiments conducted in the zebrafish model of TB, immunization with α-Gal followed by experimental infection with α-Gal-positive *M. marinum* resulted in protective responses that could be associated with TRAIM-mediated mechanisms [121]. While α-Gal present in mycobacteria may antagonize TLR2-mediated immune response, immunization with this glycan resulted in antibody-mediated interference with the mycobacterial antagonistic effect to promote TLR/NF-κB/AKR-mediated immune response and upregulation of pro-inflammatory cytokines [121]. One of the questions that arise from these results is whether ticks can induce a TRAIM response in α-Gal-negative hosts. Despite the growing incidence of AGS, only a small fraction of the individuals exposed to tick bites develop this syndrome [123]. Recently, a model for the study of the AGS was established in zebrafish [124]. Although the immune mechanisms associated with AGS
<figure><img src="image_11.png" /></figure>

have not been fully elucidated, results in zebrafish treated
with tick saliva showed the activation of innate immune
responses mediated by upregulation of C3 in fish intes-
tine [124]. If true in humans, the hypothesis is that α-Gal
and other unknown tick salivary biomolecules induce
TRAIM through C3 and other mechanisms, which may
protect against AGS and tick-borne and non-tick-borne
pathogens such as mycobacteria. This hypothesis may be
addressed by a better characterization of the immune
mechanisms induced by tick saliva and in the GalT-KO
mouse and zebrafish animal models.

COOPERATION IN PATHOGEN-
HOST INTERACTIONS

Tick-host-pathogen interactions evolved as conflict and cooperation [117, 123]. For mycobacteria, TB represents a clear conflict in host-pathogen interactions, but is there any cooperation? The exposure to live BCG vaccine and to HIMB has been shown to train the innate immune system for TRAIM-mediated protection to other pathogens [7]. These results support that *M. tuberculosis* complex bacteria can trigger this type of response. Therefore, it should not be ruled out that natural exposure to these species induces a TRAIM-mediated protection if occurring in the right circumstances. The high rate of latent to clinical TB would be highly suggestive of a cooperative effect of mycobacterial infections that in the long term would be beneficial, at the cost of being clearly conflicting with a few individuals with their TRAIM capabilities genetically or temporarily diminished. The ongoing coronavirus disease 19 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has challenged our knowledge of the host immune response to coronaviruses, showing an unusual immunopathology that has become the cause of this infection lethality [125]. Therefore, the characterization of the immunological mechanisms involved in COVID-19 symptoms and protective response is important to advance in disease prevention and control. Recently, a possible protective effect against SARS-CoV-2 infection elicited by β-glucans and α-Gal glycan has been postulated [10, 126]. Proposed protective mechanisms elicited by these biomolecules include TRAIM [10] and macrophage response, complement system, and upregulation of pro-inflammatory cytokines through the TLR2/NF-κB innate immune pathway [126].

It has also been suggested that BCG vaccination con-
tributes to modulate morbidity and mortality by COVID-
19 [67, 123, 127, 128]. Accordingly, despite other factors
such as blood group type distribution that affect the pro-
tective antibody response to α-Gal [129], the lower

prevalence of COVID-19 in most African countries may
be due (in addition to under-recording of cases) to BCG-
induced TRAIM [18, 130] in conjunction with other host
factors that may also reduce COVID-19 symptomatology
and mortality [131]. Moreover, it cannot be ignored that
TB itself may both induce TRAIM and selectively build
up an immunologically stronger population [132]. This
selective pressure along with others with a bottleneck
effect like plague might have been critical for allowing
the dense concentration of individuals of the urban way
of life of large human communities [133]. Therefore, the
study of the innate immune and TRAIM response to live
or inactivated mycobacteria and their components
deserves attention to better characterize the evolution of
host-pathogen cooperation.

ZEBRAFISH: A SUITABLE ANIMAL
MODEL FOR THE STUDY OF TRAIM

Traditionally, mammalian models and especially mice, have been used in immunology research. In the last few decades, fish models have arisen as an attractive alternative [134–136]. Namely, the zebrafish (*D. rerio*) presents numerous advantages as animal model, such as small size, rapid life cycle and translucency of embryos, as well as sharing high genomic homology with humans [137, 138]. The major counterparts of the mammalian innate immune system, such as macrophages and neutrophiles [139], as well as cytokines, TLRs and nucleotide-binding oligomerization domain-like receptors (NLRs), have been identified in zebrafish [135, 138, 140]. For that matter, the zebrafish model has been consolidated as a unique tool to assess macrophages and neutrophils, which are, indeed, main phagocytic cells of the innate system [141]. In a series of bacterial challenge experiments in zebrafish embryos, Herbomel et al. [142] demonstrated that early macrophages are able to phagocytose *Escherichia coli* and *Bacillus subtilis* in both blood and body cavities, attaining an efficient control of bacterial infection the absence of lymphocytes. As in mammals, the entire macrophage population displayed an activated state, despite the fact that only a fraction migrated to the infection site. Likewise, zebrafish macrophages present pathogen recognition receptors in the cell surface resembling the mannose receptor of mammalian resident macrophage [142].

Over the past few decades, the zebrafish model has
been extensively applied in fungal [143], bacterial [144]
and viral [145] infection studies. Furthermore, TRAIM
has been reported in teleost fishes [7, 146–149], including
zebrafish [150]. For instance, haematopoietic stem cell
expansion and emergency granulopoiesis induction
occurred after infection with *Salmonella* [151] and
*Shigella*, respectively, in zebrafish [152]. Likewise, zebrafish primed with live or heat-killed *Salmonella typhimurium* survived better to subsequent infection with *Streptococcus iniae* than non-primed and infected fishes [153]. Moreover, correlation between several innate multigene families and phenotype of zebrafish surviving a rhabdovirus infection has been described [154].

In aquaculture, immunostimulants have been adminis-
tered to fish directly via feed pellets (oral immunization) or
indirectly via bath treatment (mucosal immunization) [104,
147]. Even though various immunostimulants have been
tested in fish, β-glucans are by far the most used in aquacul-
ture [155]. β-glucans have been demonstrated to induce
TRAIM in several fish species, eliciting protection against
pathogen infection [7, 156–158]. Indeed, zebrafish primed
with β-glucans prior to infection with spring viremia of carp
virus (SVCV) [159] or *Salmonella typhimurium* [153]
improved survival and increased expression of genes
involved in the innate immune response compared with
non-primed and infected fishes.

Although not to such a great extent, the protective
effect of mycobacteria has also been explored in fish
models. For instance, an increased bacteriolytic activity
of the serum in Japanese flounder (*Paralichthys olivaceus*)
challenged with *Nocardia* was observed upon vaccination
with BCG [160]. Moreover, several experiments of immu-
nization with HIMB or α-Gal conducted in zebrafish
attributed the protective effect of the immunostimulant
against mycobacterial infection to the stimulation of
innate response [99, 104, 121, 161]. In fact, protection
correlated with the upregulation of innate components
involved in immunity against mycobacteria such as the
complement component C3 and the pro-inflammatory
cytokine IL-1β [162]. This protective innate response was
mediated by TLR activation of the Nuclear Factor kappa-
light-chain-enhancer of activated B cells (NF-κB)/AKR2
pathway [99, 104, 121] and, thus, revealed immunological
mechanisms compatible with TRAIM.

Ultimately, the *M. marinum*-zebrafish model has
emerged as a validated tool for the research of TB patho-
geny, diagnosis and treatment [163]. Overall, studies
administrating live (BCG) or inactivated (HIMB) *M. bovis*
to zebrafish via parenteral, oral or mucosal routes sup-
port the use of the species as an animal model to deepen
in the knowledge of TRAIM-mediated mechanisms in
mycobacteria-host interactions [104, 154, 164–167].

CONCLUSIONS AND FUTURE
DIRECTIONS

Exposure to live microbes or microbe-derived products
such as heat-inactivated cells can train the innate

immune system for TRAIM-mediated responses to sec-
ondary stimuli. In particular, immunization with HIMB
or α-Gal elicits protective responses against several patho-
gens such as *Mycobacterium*, *Salmonella*, *Plasmodium*
and *Leishmania* in different hosts. These observations
point out to macrophage lytic capabilities and cytokine
regulation as two key components in non-specific innate
immune responses against bacterial infections. These
mechanisms could be surrogates of TRAIM-mediated
protection indicative of host response when antigen-
specific immune responses are not effective [59]. Also,
the study of the TRAIM response induced by mycobac-
teria deserves attention to better characterize the evolu-
tion of host-pathogen cooperation both for identifying
the aetiology of some diseases and for finding new thera-
peutic strategies. Use of zebrafish provides a convenient
complete biological system that could help to carry out
this research.

AUTHOR CONTRIBUTIONS

*Prepared the original draft:* José de la Fuente, Ramón A. Juste, Elisa Ferreras-Colino, María A. Risalde and Christian Gortázar. *Contributed to review and editing:* all authors. *Project administration and funding acquisition:* María A. Risalde and Christian Gortázar. All authors have read and agreed to the published version of the manuscript.

ACKNOWLEDGEMENTS

This study was funded by Junta de Comunidades de Castilla-La Mancha, Spain, and EU-FEDER, projects MYCOTRAINING SBPLY/19/180501/000174 and CCM17-PIC-036 (SBPLY/17/180501/000185), Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación MCIN/AEI/10.13039/501100011033, Spain and EU-FEDER (Grant BIO-GAL PID2020-116761GB-I00) and by Principado de Asturias, PCTI 2021-2023 (GRUPIN: IDI2021-000102) and FEDER. EFC holds a PhD contract from the UCLM co-supported by the European Social Fund (2020/3836).

DATA AVAILABILITY STATEMENT

Data sharing not applicable to this article as no datasets
were generated or analysed during the current study.

ORCID

Elisa Ferreras-Colino https://orcid.org/0000-0002-7286-8760

REFERENCES

1. Varadé J, Magadán S, González-Fernández Á. Human immunology and immunotherapy: main achievements and challenges. Cell Mol Immunol. 2020;18(4):805-28.

2. Kaufmann SHE. Immunology's coming of age. Front Immunol. 2019;10:684.
<figure><img src="image_12.png" /></figure>

3. Netea MG, Quintin J, van der Meer JWM. Trained immunity: a memory for innate host defense. Cell Host Microbe. 2011; 9(5):355-61. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21575907

4. Netea MG, Domínguez-Andrés J, Barreiro LB, Chavakis T, Divangahi M, Fuchs E, et al. Defining trained immunity and its role in health and disease. Nat Rev Immunol. 2020;20(6): 375-88. https://doi.org/10.1038/s41577-020-0285-6

5. Saeed S, Quintin J, Kerstens HHD, Rao NA, Aghajanirefah A, Matarese F, et al. Epigenetic programming of monocyte-to-macrophage differentiation and trained innate immunity. Science (80-). 2014;345(6204):1251086.

6. Domínguez-Andrés J, Fanucchi S, Joosten LAB, Mhlanga MM, Netea MG. Advances in understanding molecular regulation of innate immune memory. Curr Opin Cell Biol. 2020;63:68-75. https://doi.org/10.1016/j.ceb.2019.12.006

7. Byrne KA, Loving CL, McGill JL. Innate immunomodulation in food animals: evidence for trained immunity? Front Immunol. 2020;11:1-15. Available from: www.frontiersin.org

8. Cavaillon J-M, Pitton C, Fitting C. Endotoxin tolerance is not a LPS-specific phenomenon: partial mimicry with IL-1, IL-10 and TGFβ. J Endotoxin Res. 1994;1(1):21-9.

9. Foster SL, Hargreaves DC, Medzhitov R. Gene-specific control of inflammation by TLR-induced chromatin modifications. Nature. 2007;447(7147):972-8.

10. Geller A, Yan J. Could the induction of trained immunity by β-Glucan serve as a defense against COVID-19? Front Immunol. 2020;11:1782.

11. Rusek P, Wala M, Druszczyńska M, Fol M. Infectious agents as stimuli of trained innate immunity. Int J Mol Sci. 2018; 19(2):456.

12. Netea MG, Giamarellos-Bourboulis EJ, Domínguez-Andrés J, Curtis N, van Crevel R, van de Veerdonk FL, et al. Trained immunity: a tool for reducing susceptibility to and the severity of SARS-CoV-2 infection. Cell. 2020;181(5):969-77.

13. Elberg SS, Schneider P, Fong J. Cross-immunity between *Brucella melitensis* and *Mycobacterium tuberculosis*; intracellular behavior of *Brucella melitensis* in monocytes from vaccinated animals. J Exp Med. 1957;106(4):545-54.

14. Mackaness GB. The immunological basis of acquired cellular resistance. J Exp Med. 1964;120(1):105-20.

15. Allison AC. Macrophage activation and non-specific immunity. Int Rev Exp Pathol. 1978;77:303-46.

16. Aaby P, Benn CS. Developing the concept of beneficial non-specific effect of live vaccines with epidemiological studies. Clin Microbiol Infect. 2019;25(12):1459-67. Available from: https://www.ncbi.nlm.nih.gov/pubmed/31449870

17. Aaby P, Benn CS. Stopping live vaccines after disease eradication may increase mortality. Vaccine. 2020;38(1):10-4. https://doi.org/10.1016/j.vaccine.2019.10.034

18. Covián C, Fernández-Fierro A, Retamal-Díaz A, Díaz FE, Vasquez AE, Lay MK, et al. BCG-induced cross-protection and development of trained immunity: implication for vaccine design. Front Immunol. 2019;10:1-14.

19. Shann F. Nonspecific effects of vaccines and the reduction of mortality in children. Clin Ther. 2013;35(2):109-14.

20. Finch CE. Evolution of the human lifespan and diseases of aging: roles of infection, inflammation, and nutrition. Proc Natl Acad Sci U S A. 2010;107(Suppl 1):1718-24.

21. Villagra C, Frías-Lasserre D. Epigenetic molecular mechanisms in insects. Neotrop Entomol. 2020;49:615-42. https://doi.org/10.1007/s13744-020-00777-8

22. Mukherjee K, Dubovskiy I, Grizanova E, Lehmann R, Vilcinskas A. Epigenetic mechanisms mediate the experimental evolution of resistance against parasitic fungi in the greater wax moth *Galleria mellonella*. Sci Rep. 2019;9(1):1626.

23. Harpur BA, Zayed A. Accelerated evolution of innate immunity proteins in social insects: adaptive evolution or relaxed constraint? Mol Biol Evol. 2013;30(7):1665-74. Available from: https://academic.oup.com/mbe/article/30/7/1665/971880

24. Chan QWT, Melathopoulos AP, Pernal SF, Foster LJ. The innate immune and systemic response in honey bees to a bacterial pathogen, *Paenibacillus larvae*. BMC Genomics. 2009;21:10.

25. Kumar A, Srivastava P, Sirisena P, Dubey SK, Kumar Id R, Sunil S. Mosquito Innate Immunity. Insects. 2018;9(95):1-34. Available from: www.mdpi.com/journal/insects

26. Sharrock J, Sun JC. Innate immunological memory: from plants to animals. Curr Opin Immunol. 2020;62:69-78.

27. Gordon S. Phagocytosis: an immunobiologic process. Immunity. 2016;44(3):463-75. https://doi.org/10.1016/j.immuni.2016.02.026

28. Sica A, Mantovani A. Macrophage plasticity and polarization: in vivo veritas. J Clin Investig. 2012;122:787-95.

29. Hoeksema MA, De Winther MPJ. Epigenetic regulation of monocyte and macrophage function. Antioxid Redox Signal. 2016;25(14):758-74.

30. Katzmarski N, Domínguez-Andrés J, Cirovic B, Renieris G, Ciarlo E, Le Roy D, et al. Reply to: 'Lack of evidence for intergenerational inheritance of immune resistance to infections.' Nat Immunol. 2022;23(2):208-9.

31. Katzmarski N, Domínguez-Andrés J, Cirovic B, Renieris G, Ciarlo E, Le Roy D, et al. Transmission of trained immunity and heterologous resistance to infections across generations. Nat Immunol. 2021;22(11):1382-90. Available from: https://pubmed.ncbi.nlm.nih.gov/34663978/

32. Van Der Heijden CDCC, Noz MP, Joosten LAB, Netea MG, Riksen NP, Keating ST. Epigenetics and trained immunity. Antioxid Redox Signal. 2018;29(11):1023-40. Available from: https://www.liebertpub.com/doi/abs/10.1089/ars.2017.7310

33. Fanucchi S, Fok ET, Dalla E, Shibayama Y, Börner K, Chang EY, et al. Immune genes are primed for robust transcription by proximal long noncoding RNAs located in nuclear compartments. Nat Genet. 2019;51:138-50. https://doi.org/10.1038/s41588-018-0298-2

34. Riksen NP, Netea MG. Immunometabolic control of trained immunity. Mol Aspects Med. 2020;77:100897. https://doi.org/10.1016/j.mam.2020.100897

35. Saeed S, Quintin J, Kerstens HHD, Rao NA, Aghajanirefah A, Matarese F, et al. Epigenetic programming during monocyte to macrophage differentiation and trained innate immunity. Science. 2014;345(6204):1251086.

36. Arts RJW, Novakovic B, ter Horst R, Carvalho A, Bekkering S, Lachmandas E, et al. Glutaminolysis and fumarate accumulation integrate immunometabolic and epigenetic programs in trained immunity. Cell Metab. 2016;24(6):807-19.

37. Keating ST, Groh L, van der Heijden CDCC, Rodriguez H, dos Santos JC, Fanucchi S, et al. The Set7 lysine methyltransferase regulates plasticity in oxidative phosphorylation necessary for
trained immunity induced by β-Glucan. Cell Rep. 2020;31(3): 107548.

38. Gordon S. Phagocytosis: the legacy of Metchnikoff. Cell. 2016; 166(5):1065-8. https://doi.org/10.1016/j.cell.2016.08.017

39. Adams DO. The granulomatous inflammatory response: a review. J Am Pathol. 1976;84(1):164-91.

40. Barbieri TA, Correa WM. Human macrophage culture: the lep-rosy prognostic test (LPT). Int J Lepr. 1967;35(3):377-81.

41. Mills CD, Kincaid K, Alt JM, Heilman MJ, Hill AM. M-1/M-2 macrophages and the Th1/Th2 paradigm. J Immunol. 2000; 164(12):6166-73. Available from: https://pubmed.ncbi.nlm.nih.gov/10843666/

42. Liu YC, Zou XB, Chai YF, Yao YM. Macrophage polarization in inflammatory diseases. Int J Biol Sci. 2014;10(5):520-9.

43. Yunna C, Mengru H, Lei W, Weidong C. Macrophage M1/M2 polarization. Eur J Pharmacol. 2020;15:877.

44. Jayasingam SD, Citartan M, Thang TH, Mat Zin AA, Ang KC, Ch'ng ES. Evaluating the polarization of tumor-associated macrophages into M1 and M2 phenotypes in human cancer tissue: technicalities and challenges in routine clinical practice. Front Oncol. 2020;9:1512.

45. Murray PJ, Allen JE, Biswas SK, Fisher EA, Gilroy DW, Goerdt S, et al. Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity. 2014; 41(1):14-20.

46. Miedema F, Tersmette M, van Lier RAW. AIDS pathogenesis: a dynamic interaction between HIV and the immune system. Immunol Today. 1990;11:293-7.

47. Blok BA, Arts RJW, van Crevel R, Benn CS, Netea MG. Trained innate immunity as underlying mechanism for the long-term, nonspecific effects of vaccines. J Leukoc Biol. 2015; 98(3):347-56. Available from: https://pubmed.ncbi.nlm.nih.gov/26150551/

48. Artigas-Jerónimo S, Villar M, Cabezas-Cruz A, Valdés JJ, Estrada-Peña A, Alberdi P, et al. Functional evolution of subo-lesin/akirin. Front Physiol. 2018;9:1612.

49. Janeway C, Travers P, Walport M. Immunobiology – NCBI bookshelf. New York: Garland Science; 2001. Available from: https://www.ncbi.nlm.nih.gov/books/NBK10757/

50. Polanowska J, Chen JX, Soulé J, Omi S, Belougne J, Taffoni C, et al. Evolutionary plasticity in the innate immune function of Akirin. PLoS Genet. 2018;14(7):e1007494.

51. Huang Z, Luo Q, Guo Y, Chen J, Xiong G, Peng Y, et al. *Mycobacterium tuberculosis*-induced polarization of human macrophage orchestrates the formation and development of tuberculous granulomas in vitro. PLoS One. 2015;10(6):e0129744.

52. Ilg T. Investigations on the molecular mode of action of the novel immunostimulator ZelNate: activation of the cGAS-STING pathway in mammalian cells. Mol Immunol. 2017;90:182-9.

53. Ifrim DC, Quintin J, Joosten LAB, Jacobs C, Jansen T, Jacobs L, et al. Trained immunity or tolerance: opposing functional programs induced in human monocytes after engagement of various pattern recognition receptors. Clin Vaccine Immunol. 2014;21(4):534-45. Available from: https://pubmed.ncbi.nlm.nih.gov/24521784/

54. Arteche-Villasol N, Gutiérrez-Expósito D, Vallejo R, Espinosa J, Elguezabal N, Ladero-Auñon I, et al. Early response of monocyte-derived macrophages from vaccinated and non-vaccinated goats against in vitro infection with *Mycobacterium*

avium subsp. paratuberculosis. Vet Res. 2021;52(1):69. https://doi.org/10.1186/s13567-021-00940-y

55. Hu Z, Lu S-H, Lowrie DB, Fan X-Y. Trained immunity: a Yin-Yang balance. MedComm. 2022;3(1):e121. Available from: http://www.ncbi.nlm.nih.gov/pubmed/35281787

56. Luca S, Mihaescu T. History of BCG vaccine. Maedica. 2013;8(1): 53-8. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24023600

57. World Health Organization. BCG vaccine: WHO position paper, February 2018 – recommendations. Vaccine. 2018; 36(24):3408-10. Available from: http://www.ncbi.nlm.nih.gov/pubmed/29609965

58. Trunz BB, Fine P, Dye C. Effect of BCG vaccination on childhood tuberculous meningitis and military tuberculosis worldwide: a meta-analysis and assessment of cost-effectiveness. Lancet. 2006;367(9517):1173-80. Available from: http://www.thelancet.com/article/S0140673606685073/fulltext

59. Butkeviciute E, Jones CE, Smith SG. Heterologous effects of infant BCG vaccination: potential mechanisms of immunity. Future Microbiol. 2018;13(10):1193-208.

60. Aaby P, Roth A, Ravn H, Mutna Napirna B, Rodrigues A, Lisse IM, et al. Randomized trial of BCG vaccination at birth to low-birth-weight children: beneficial nonspecific effects in the neonatal period? J Infect Dis. 2011;204:245-52. Available from: https://academic.oup.com/jid/article/204/2/245/833883

61. De Castro MJ, Pardo-Seco J, Martinón-Torres F. Nonspecific (heterologous) protection of neonatal BCG vaccination against hospitalization due to respiratory infection and sepsis. Clin Infect Dis. 2015;60(11):1611-9. Available from: https://academic.oup.com/cid/article/60/11/1611/356084

62. Howard JG, Biozzi G, Halpern BN, Stifel C, Mouton D. The effect of *Mycobacterium tuberculosis* (BCG) infection on the resistance of mice to bacterial endotoxin and *Salmonella enteritidis* infection. Br J Exp Pathol. 1959;40(3):281-90. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2083463/

63. Kleinnijenhuis J, van Crevel R, Netea MG. Trained immunity: consequences for the heterologous effects of BCG vaccination. Trans R Soc Trop Med Hyg. 2015;109(1):29-35. Available from: https://academic.oup.com/trstmh/article-lookup/doi/10.1093/trstmh/tru168

64. Kleinnijenhuis J, Quintin J, Preijers F, Benn CS, Joosten LAB, Jacobs C, et al. Long-lasting effects of BCG vaccination on both heterologous Th1/Th17 responses and innate trained immunity. J Innate Immun. 2014;6:152-8. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24192057

65. Novakovic B, Habibi E, Wang S-Y, Arts RJW, Davar R, Megchelenbrink W, et al. β-Glucan reverses the epigenetic state of LPS-induced immunological tolerance. Cell. 2016; 167(5):1354-68. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0092867416313162

66. Moorlag SJCFM, Arts RJW, van Crevel R, Netea MG. Non-specific effects of BCG vaccine on viral infections. Clin Microbiol Infect. 2019;25(12):1473-8.

67. Moorlag SJCFM, van Deuren RC, van Werkhoven CH, Jaeger M, Debisarun P, Taks E, et al. Safety and COVID-19 symptoms in individuals recently vaccinated with BCG: a retrospective cohort study. Cell Rep Med. 2020;1(5):100073.
68. Guerra-Maupome M, Vang DX, McGill JL. Aerosol vaccination with Bacille Calmette-Guerin induces a trained innate immune phenotype in calves. PLoS One. 2019;14(2):e1-16.

69. Brook B, Harbeson DJ, Shannon CP, Cai B, He D, Ben-Othman R, et al. BCG vaccination-induced emergency granulopoiesis provides rapid protection from neonatal sepsis. Sci Transl Med. 2020;12(542):eaax4517. Available from: https://doi.org.sciencemag.org/lookup/doi/10.1126/scitranslmed.aax4517

70. Byrne KA, Tuggle CK, Loving CL. Differential induction of innate memory in porcine monocytes by β-glucan or bacillus Calmette-Guerin. Innate Immun. 2021;27:448-60.

71. Koeken VACM, Charlotte L, Mourits VP, Moorlag SJCFM, Walk J, Cirovic B, et al. BCG vaccination in humans inhibits systemic inflammation in a sex-dependent manner. J Clin Invest. 2020;130(10):5591-602.

72. Floc'h F, Werner GH. Increased resistance to virus infections of mice inoculated with BCG (Bacillus Calmette Guerin). Ann Immunol. 1976;127(2):173-86. Available from: https://pubmed.ncbi.nlm.nih.gov/180868/

73. Starr S, Visintine A, Tomeh M, Nahmias A. Effects of immunostimulants on resistance of newborn mice to herpes simplex type 2 infection. Proc Soc Exp Biol Med. 1976;152(1):57-60. Available from: https://pubmed.ncbi.nlm.nih.gov/177992/

74. Spencer JC, Ganguly R, Waldman RH. Nonspecific protection of mice against influenza virus infection by local or systemic immunization with Bacille Calmette-Guerin. J Infect Dis. 1977;136(2):171-5. Available from: https://academic.oup.com/jid/article/136/2/171/909468

75. Mukherjee S, Subramaniam R, Chen H, Smith A, Keshava S, Shams H. Boosting efferocytosis in alveolar space using BCG vaccine to protect host against influenza pneumonia. PLoS One. 2017;12(7):e0180143.

76. Dubos RJ, Schaedler RW. Effects of cellular constituents of mycobacteria on the resistance of mice to heterologous infections: I. Protective effects. J Exp Med. 1957;106(5):703-17.

77. Fortier AH, Mock BA, Meltzer MS, Nacy CA. *Mycobacterium bovis* BCG-induced protection against cutaneous and systemic Leishmania major infections of mice. Infect Immun. 1987; 55(7):1707-14.

78. Parra M, Liu X, Derrick SC, Yang A, Tian J, Kolibab K, et al. Molecular analysis of non-specific protection against murine malaria induced by BCG vaccination. PLoS One. 2013;8(7):e1-8.

79. Ortiz-Ortiz L, Gonzalez-Mendoza A, Lamoyi E. A vaccination procedure against *Trypanosoma cruzi* infection in mice by nonspecific immunization. J Immunol. 1975;114(4):1424-5.

80. Clark I, Allison A, Cox F. Protection of mice against *Babesia* and *Plasmodium* with BCG. Nature. 1976;259(5541):309-11. Available from: https://pubmed.ncbi.nlm.nih.gov/765838/

81. Kleinnijenhuis J, Quintin J, Preijers F, Joosten LAB, Ifrim DC, Saeed S, et al. Bacille Calmette-Guerin induces NOD2-dependent nonspecific protection from reinfection via epigenetic reprogramming of monocytes. Proc Natl Acad Sci U S A. 2012;109(43):17537-42.

82. Arts RJW, Moorlag SJCFM, Novakovic B, Li Y, Wang SY, Oosting M, et al. BCG vaccination protects against experimental viral infection in humans through the induction of cytokines associated with trained immunity. Cell Host Microbe. 2018;23(1):89-100. https://doi.org/10.1016/j.chom.2017.12.010

83. Salem A, Nofal A, Hosny D. Treatment of common and plane warts in children with topical viable Bacillus Calmette-Guerin. Pediatr Dermatol. 2013;30(1):60-3. Available from: https://pubmed.ncbi.nlm.nih.gov/22958215/

84. Walk J, de Bree LCJ, Graumans W, Stoter R, van Gemert GJ, van de Vegte-Bolmer M, et al. Outcomes of controlled human malaria infection after BCG vaccination. Nat Commun. 2019;10(1):874. https://doi.org/10.1038/s41467-019-08659-3

85. Netea MG, van Crevel R. BCG-induced protection: effects on innate immune memory. Semin Immunol. 2014;26(6):512-7. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1044532314000888

86. Liu H, Li P, Wei Z, Zhang C, Xia M, Du Q, et al. Regulation of T cell differentiation and function by epigenetic modification enzymes. Semin Immunopathol. 2019;41(3):315-26. Available from: https://link.springer.com/article/10.1007/s00281-019-00731-w

87. Arts RJW, Carvalho A, La Rocca C, Palma C, Rodrigues F, Silvestre R, et al. Immunometabolic pathways in BCG-induced trained immunity. Cell Rep. 2016;17(10):2562-71.

88. Saroha M, Faridi M, Batra P, Kaur I, Dewan D. Immunogenicity and safety of early vs delayed BCG vaccination in moderately preterm (31-33 weeks) infants. Hum Vaccines Immunother. 2015;11(12):2864.

89. Buddle BM, Vordermeier HM, Chambers MA, de Klerk-Lorist LM. Efficacy and safety of BCG vaccine for control of tuberculosis in domestic livestock and wildlife. Front Vet Sci. 2018;5:259.

90. Marciano B, Huang C, Joshi G, Rezaei N, Carvalho B, Allwood Z, et al. BCG vaccination in SCID patients: complications, risks and vaccination policies. J Allergy Clin Immunol. 2014;133(4):1134-41. Available from: www.who.int/tb/publications/global_report/gtbr12_main.pdf

91. Beltrán-Beck B, De La Fuente J, Garrido JM, Aranaz A, Sevilla I, Villar M, et al. Oral vaccination with heat inactivated *Mycobacterium bovis* activates the complement system to protect against tuberculosis. PLoS One. 2014;9(5):e98048.

92. Angelidou A, Levy O, van Haren S, Angelidou A, Diray-Arce J, Giulia Conti M, et al. BCG as a case study for precision vaccine development: lessons from vaccine heterogeneity, trained immunity, and immune ontogeny. Front Microbiol. 2020;1:332. Available from: www.frontiersin.org

93. Garrido JM, Sevilla IA, Beltrán-Beck B, Minguijón E, Ballesteros C, Galindo RC, et al. Protection against tuberculosis in eurasian wild boar vaccinated with heat-inactivated *Mycobacterium bovis*. PLoS One. 2011;6(9):e24905.

94. Jones GJ, Steinbach S, Sevilla IA, Garrido JM, Juste R, Vordermeier HM. Oral vaccination of cattle with heat inactivated *Mycobacterium bovis* does not compromise bovine TB diagnostic tests. Vet Immunol Immunopathol. 2016;182:85-8.

95. Roy A, Risalde MA, Casal C, Romero B, de Juan L, Menshawy AM, et al. Oral vaccination with heat-inactivated *Mycobacterium bovis* does not interfere with the Antemortem diagnostic techniques for tuberculosis in goats. Front Vet Sci. 2017;4:124.

96. Beltrán-Beck B, Romero B, Boadella M, Casal C, Bezos J, Mazariegos M, et al. Tonsils of the soft palate do not mediate the response of pigs to oral vaccination with heat-inactivated *Mycobacterium bovis*. Clin Vaccine Immunol. 2014;21(8):1128-36.
97. Thomas J, Risalde MÁ, Serrano M, Sevilla I, Geijo M, Ortiz JA, et al. The response of red deer to oral administration of heat-inactivated *Mycobacterium bovis* and challenge with a field strain. Vet Microbiol. 2017;208:195-202.

98. Balseiro A, Prieto JM, Álvarez V, Lesellier S, Davé D, Salguero FJ, et al. Protective effect of oral BCG and inactivated *Mycobacterium bovis* vaccines in European Badgers (*Meles meles*) experimentally infected with *M. bovis*. Front Vet Sci. 2020 Feb;7:41. Available from: https://www.frontiersin.org/articles/10.3389/fvets.2020.00041/full

99. López V, Risalde MA, Contreras M, Mateos-Hernández L, Vicente J, Gortázar C, et al. Heat-inactivated *Mycobacterium bovis* protects zebrafish against mycobacteriosis. J Fish Dis. 2018;41(10):1515-28.

100. Naranjo V, Ayoubi P, Vicente J, Ruiz-Fons F, Gortazar C, Kocan KM, et al. Characterization of selected genes upregulated in non-tuberculous European wild boar as possible correlates of resistance to *Mycobacterium bovis* infection. Vet Microbiol. 2006 Aug;116(1-3):224-31.

101. Juste RA, Alonso-Hearn M, Garrido JM, Abendaño N, Sevilla IA, Gortazar C, et al. Increased lytic efficiency of bovine macrophages trained with killed mycobacteria. PLoS One. 2016;11(11):e0165607.

102. Juste Jordán RA, Domínguez Rodríguez L, Gortázar Schmidt C, De La Fuente García J de J, Garrido Urkullu JM, Agirregomoskorta Sevilla I, et al. Immunostimulant for use against pathogens. 2020.

103. Vaz-Rodrigues R, Ferreras-Colino E, Ugarte-Ruíz M, Pesciaroli M, Thomas J, García-Seco T, et al. Nonspecific protection of heat-inactivated *Mycobacterium bovis* against salmonella Choleraesuis infection in pigs. Vet Res. 2022;53(1):31.

104. Risalde MA, López V, Contreras M, Mateos-Hernández L, Gortázar C, de la Fuente J. Control of mycobacteriosis in zebrafish (*Danio rerio*) mucosally vaccinated with heat-inactivated *Mycobacterium bovis*. Vaccine. 2018;36(30):4447-53.

105. Juste R, Domínguez L, Gortázar C, De La Fuente J, Garrido J, Sevilla I, et al. Immunostimulant for use against pathogens. Madrid, Spain; PCT/EP2019/083730; 2018. p. 50.

106. Kawakami H, Shinohara N, Sakai M. The non-specific immunostimulation and adjuvant effects of vibrio anguillarum Bacterin, M-glucan, chitin and Freund's complete adjuvant against Pasteurella piscicida infection in yellowtail. Fish Pathol. 1998;33(4):287-92.

107. Contreras M, Kasaija PD, Merino O, De La Cruz-Hernandez NI, Gortazar C, De La Fuente J. Oral vaccination with a formulation combining rhipicephalus microplus subolesin with heat inactivated *Mycobacterium bovis* reduces tick infestations in cattle. Front Cell Infect Microbiol. 2019;9:45.

108. De La Fuente J, Contreras M. Tick vaccines: current status and future directions. Expert Rev Vaccines. 2015;14:1367-76.

109. Huang JH, Lin CY, Wu SY, Chen WY, Chu CL, Brown GD, et al. CR3 and Dectin-1 collaborate in macrophage cytokine response through association on lipid rafts and activation of Syk-JNK-AP-1 pathway. PLoS Pathog. 2015;11(7):e1004985.

110. Rabinovich GA, Kooyk Y v, Cobb BA. Glycobiology of immune responses. Ann N Y Acad Sci. 2012;1253(1):1-15.

111. Di Luzio NR, Williams DL. Protective effect of glucan against systemic *Staphylococcus aureus* septicemia in normal and leukemic mice. Infect Immun. 1978;20(3):804-10.

112. Bistoni F, Verducci G, Perito S, Vecchiarelli A, Puccetti P, Marconi P, et al. Immunomodulation by a low-virulence, agerminative variant of *Candida albicans*. Further evidence for macrophage activation as one of the effector mechanisms of nonspecific anti-infectious protection. J Med Vet Mycol. 1988; 26(5):285-99.

113. Quintin J, Saeed S, Martens JHA, Giamarellos-Bourboulis EJ, Ifrim DC, Logie C, et al. *Candida albicans* infection affords protection against reinfection via functional reprogramming of monocytes. Cell Host Microbe. 2012;12(2):223-32.

114. Bistoni F, Vecchiarelli A, Cenci E, Puccetti P, Marconi P, Cassone A. Evidence for macrophage-mediated protection against lethal *Candida albicans* infection. Infect Immun. 1986; 51(2):668-74.

115. Cheng SC, Quintin J, Cramer RA, Shepardson KM, Saeed S, Kumar V, et al. MTOR- and HIF-1α-mediated aerobic glycolysis as metabolic basis for trained immunity. Science (80- ). 2014;345(6204):1250684.

116. Cabezas-Cruz A, de la Fuente J, Hodžić A, Román-Carrasco P, Mateos-Hernández L, Gerhard Duscher G, et al. Environmental and molecular drivers of the α-gal syndrome. Front Immunol. 2019;10:1210. https://doi.org/10.3389/fimmu.2019.01210

117. De La Fuente J, Pacheco I, Villar M, Cabezas-Cruz A. The alpha-Gal syndrome: new insights into the tick-host conflict and cooperation. Parasit Vectors. 2019;12:154-8. https://doi.org/10.1186/s13071-019-3413-z

118. Yilmaz B, Portugal S, Tran TM, Gozzelino R, Ramos S, Gomes J, et al. Gut microbiota elicits a protective immune response against malaria transmission. Cell. 2014;159(6):1277-89. Available from: http://www.cell.com/article/S00928674104014251/fulltext

119. Galili U. Evolution in primates by "catastrophic-selection" interplay between enveloped virus epidemics, mutated genes of enzymes synthesizing carbohydrate antigens, and natural anti-carbohydrate antibodies. Am J Phys Anthropol. 2019;168: 352-63.

120. Mateos-Hernández L, Risco-Castillo V, Torres-Maravilla E, Bermúdez-Humarán LG, Alberdi P, Hodžić AH, et al. Gut microbiota abrogates anti-α-gal IgA response in lungs and protects against experimental *Aspergillus* infection in poultry. 2020;8:285. Available from: www.mdpi.com/journal/vaccines

121. Pacheco I, Contreras M, Villar M, Risalde MA, Alberdi P, Cabezas-Cruz A, et al. Vaccination with alpha-Gal protects against mycobacterial infection in the zebrafish model of tuberculosis. Vaccines. 2020;8(2):195. https://doi.org/10.3390/vaccines8020195

122. Hodžić A, Mateos-Hernández L, de la Fuente J, Cabezas-Cruz A. α-Gal-based vaccines: advances, opportunities, and perspectives. Trends Parasitol. 2020;36(12):992-1001.

123. de la Fuente J, Villar M, Cabezas-Cruz A, Estrada-Peña A, Ayllón N, Alberdi P. Tick-host-pathogen interactions: conflict and cooperation. PLoS Pathog. 2016;12(4):e1005488.

124. Contreras M, Pacheco I, Alberdi P, Díaz-Sánchez S, Artigas-Jerónimo S, Mateos-Hernández L, et al. Allergic reactions and immunity in response to tick salivary biogenic substances and red meat consumption in the zebrafish model. Front Cell Infect Microbiol. 2020;10:78.
125. Villas-Boas GR, Rescia VC, Paes MM, Lavorato SN, de Magalhães-Filho MF, Cunha MS, et al. The new coronavirus (SARS-CoV-2): a comprehensive review on immunity and the application of bioinformatics and molecular modeling to the discovery of potential anti-SARS-CoV-2 agents. Molecules. 2020;25(18):4086.

126. Urra JM, Ferreras-Colino E, Contreras M, Cabrera C, Fernández de Mera I, Villar M, et al. The antibody response to the glycan α-gal correlates with COVID-19 disease symptoms. J Med Virol. 2021;93(4):2065-2075.

127. Jirjees FJ, Dallal Bashi YH, Al-Obaidi HJ. COVID-19 death and BCG vaccination programs worldwide. Tuberc Respir Dis. 2020;84(1):13-21.

128. Wickramasinghe D, Wickramasinghe N, Kamburugamuwa SA, Arambepola C, Samarasekera DN. Correlation between immunity from BCG and the morbidity and mortality of COVID-19. Trop Dis Travel Med Vaccines. 2020;6(1):17.

129. Cabezas-Cruz A, Mateos-Hernández L, Alberdi P, Villar M, Riveau G, Hermann E, et al. Effect of blood type on anti-α-Gal immunity and the incidence of infectious diseases. Exp Mol Med. 2017;49(3):e301.

130. O'Neill LAJ, Netea MG. BCG-induced trained immunity: can it offer protection against COVID-19? Nat Rev Immunol. 2020; 20:335-7.

131. Gortázar C, Del-Río FJR, Domínguez L, de la Fuente J. Host or pathogen-related factors in COVID-19 severity? Lancet. 2020;396:1396-7.

132. de la Fuente J, Armas O, Sánchez-Rodríguez L, Gortázar C, Lukashev AN, Almazán C, et al. Citizen science initiative points at childhood BCG vaccination as a risk factor for COVID-19. Transbound Emerg Dis. 2021;68(6):3114-9.

133. de la Fuente J, Contreras M. Vaccinomics: a future avenue for vaccine development against emerging pathogens. Expert Rev Vaccines. 2021;20(12):1561-9.

134. Yoder JA, Nielsen ME, Amemiya CT, Litman GW. Zebrafish as an immunological model system. Microbes Infect. 2002; 4(14):1469-78.

135. Trede NS, Langenau DM, Traver D, Look AT, Zon LI. The use of zebrafish to understand immunity. Immunity. 2004;20(4): 367-79.

136. Meeker ND, Trede NS. Immunology and zebrafish: spawning new models of human disease. Dev Comp Immunol. 2008; 32(7):745-57.

137. Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, Muffato M, et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature. 2013; 496(7446):498-503.

138. Stein C, Caccamo M, Laird G, Leptin M. Conservation and divergence of gene families encoding components of innate immune response systems in zebrafish. Genome Biol. 2007; 8(11):R251.

139. Rosowski EE. Determining macrophage versus neutrophil contributions to innate immunity using larval zebrafish. Dis Model Mech. 2020;13, (1):dmm041889.

140. Van Der Vaart M, Spaink HP, Meijer AH. Pathogen recognition and activation of the innate immune response in zebrafish. Adv Hematol. 2012;2012:159807.

141. Torracca V, Masud S, Spaink HP, Meijer AH. Macrophage-pathogen interactions in infectious diseases: new therapeutic

insights from the zebrafish host model. Dis Model Mech. 2014; 7(7):785-97.

142. Herbomel P, Thisse B, Thisse C. Ontogeny and behaviour of early macrophages in the zebrafish embryo. Development. 1999;126(17):3735-45.

143. Rosowski EE, Knox BP, Archambault LS, Huttenlocher A, Keller NP, Wheeler RT, et al. The zebrafish as a model host for invasive fungal infections. J Fungi. 2018;4(4):136.

144. Neely MN. The zebrafish as a model for human bacterial infections. Methods Mol Biol. 2017;1535:245-66.

145. Varela M, Figueras A, Novoa B. Modelling viral infections using zebrafish: innate immune response and antiviral research. Antiviral Res. 2017;139:59-68.

146. Petit J, Embregts CWE, Forlenza M, Wiegertjes GF. Evidence of trained immunity in a fish: conserved features in carp macrophages. J Immunol. 2019;203(1):216-24.

147. Zhang Z, Chi H, Dalmo RA. Trained innate immunity of fish is a viable approach in larval aquaculture. Front Immunol. 2019;10:42.

148. Sommerset I, Lorenzen E, Lorenzen N, Bleie H, Nerland AH. A DNA vaccine directed against a rainbow trout rhabdovirus induces early protection against a nodavirus challenge in turbot. Vaccine. 2003;21(32):4661-7.

149. Martinez-Lopez A, Garcia-Valtanen P, Ortega-Villaizan M, Chico V, Gomez-Casado E, Coll JM, et al. VHSV G glycoprotein major determinants implicated in triggering the host type I IFN antiviral response as DNA vaccine molecular adjuvants. Vaccine. 2014;32(45):6012-9.

150. Hohn C, Petrie-Hanson L. Rag1-/- mutant zebrafish demonstrate specific protection following bacterial re-exposure. PLoS One. 2012;7(9):e44451.

151. Hall CJ, Flores MV, Oehlers SH, Sanderson LE, Lam EY, Crosier KE, et al. Infection-responsive expansion of the hematopoietic stem and progenitor cell compartment in zebrafish is dependent upon inducible nitric oxide. Cell Stem Cell. 2012; 10(2):198-209.

152. Willis AR, Torracca V, Gomes MC, Shelley J, Mazon-Moya M, Filloux A, et al. *Shigella*-induced emergency granulopoiesis protects zebrafish larvae from secondary infection. MBio. 2018;9(3):e00933-18.

153. Darroch H, Astin JW, Hall CJ. Towards a new model of trained immunity: exposure to bacteria and β-glucan protects larval zebrafish against subsequent infections. Dev Comp Immunol. 2022;132:104400.

154. Estepa A, Coll J. Innate multigene family memories are implicated in the viral-survivor zebrafish phenotype. PLoS One. 2015;10(8):e0135483.

155. Anderson DP. Immunostimulants, adjuvants, and vaccine carriers in fish: applications to aquaculture. Annu Rev Fish Dis. 1992;2:281-307.

156. Álvarez-Rodríguez M, Pereiro P, Reyes-López FE, Tort L, Figueras A, Novoa B. Analysis of the long-lived responses induced by immunostimulants and their effects on a viral infection in zebrafish (*Danio rerio*). Front Immunol. 2018;9: 1575.

157. Petit J, Wiegertjes GF. Long-lived effects of administering β-glucans: indications for trained immunity in fish. Dev Comp Immunol. 2016;64:93-102. https://doi.org/10.1016/j.dci.2016.03.003
<figure><img src="image_13.png" /></figure>

158. Librán-Pérez M, Costa MM, Figueras A, Novoa B. β-Glucan administration induces metabolic changes and differential survival rates after bacterial or viral infection in turbot (*Scophthalmus maximus*). Fish Shellfish Immunol. 2018;82:173-82.

159. M. Medina-Gali R, Ortega-Villaizan M del M, Mercado L, Novoa B, Coll J, Perez L. Beta-glucan enhances the response to SVCV infection in zebrafish. Dev Comp Immunol 2018;84: 307-14.

160. Kato G, Kondo H, Aoki T, Hirono I. *Mycobacterium bovis* BCG vaccine induces non-specific immune responses in Japanese flounder against *Nocardia seriolae*. Fish Shellfish Immunol. 2012;33(2):243-50.

161. Pacheco I, Díaz-Sánchez S, Contreras M, Villar M, Cabezas-Cruz A, Gortázar C, et al. Probiotic bacteria with high alpha-gal content protect zebrafish against mycobacteriosis. Pharmaceuticals. 2021;14(7):635.

162. Benard EL, Rougeot J, Racz PI, Spaink HP, Meijer AH. Transcriptomic approaches in the zebrafish model for tuberculosis-insights into host- and pathogen-specific determinants of the innate immune response. Adv Genet. 2016;95:217-51.

163. Myllymäki H, Bäuerlein CA, Rämet M. The zebrafish breathes new life into the study of tuberculosis. Front Immunol. 2016; 7:196.

164. Cronan MR, Tobin DM. Fit for consumption: zebrafish as a model for tuberculosis. Dis Model Mech. 2014;7:777-84.

165. van Leeuwen LM, van der Sar AM, Bitter W. Animal models of tuberculosis: zebrafish. Cold Spring Harb Perspect Med. 2015;5(3):a018580.

166. López V, González-Barrio D, Lima-Barbero JF, Ortiz JA, Domínguez L, Juste R, et al. Oral administration of heat-inactivated *Mycobacterium bovis* reduces the response of farmed red deer to avian and bovine tuberculin. Vet Immunol Immunopathol. 2016;172:21-5.

167. Oksanen KE, Halfpenny NJA, Sherwood E, Harjula SKE, Hammarén MM, Ahava MJ, et al. An adult zebrafish model for preclinical tuberculosis vaccine development. Vaccine. 2013;31(45):5202-9.

**How to cite this article:** Juste RA, Ferreras-Colino E, de la Fuente J, Domínguez M, Risalde MA, Domínguez L, et al. Heat inactivated mycobacteria, alpha-Gal and zebrafish: Insights gained from experiences with two promising trained immunity inductors and a validated animal model. Immunology. 2022;167(2):139-53. https://doi.org/10.1111/imm.13529