*3.2.2 Effector functions on trained innate immune cells*

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<sup>+</sup> 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

#### **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.*

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**).

### **4. Trained immunity-based vaccines**

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 immunitybased 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**).

Following the above features, the TIbV concept can be applied to existing antiinfectious vaccines composed of microorganisms that show heterologous protection ascribed to trained immunity.

#### **Figure 2.**

*Different possibilities of trained immunity-based vaccines (TIbVs). Under the umbrella of trained immunitybased 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.*
