Vaccines are the goals of preventive medicine. So, vaccinating vulnerable populations to mitigate the harmful effects of viral diseases, such as influenza, has grown over the years. Despite these efforts, flu-vaccines are less efficient in the older adults, with effectiveness rates of 30–50% for individuals over 65 years old, compared to 70–90% for younger individuals [13]. The observed inefficacy of vaccination in this population is often attributed to the exclusion of frail subjects from clinical trials. This results in a lack of crucial data, especially regarding optimal dosing and the need for booster doses to enhance vaccine coverage and ensure effectiveness. Another key consideration for preventive vaccination is assessing the history of previous infections and vaccinations in older adults patients [14].

Regarding the causes, this mild vaccine response could be linked with the changes that the immune system undergoes during aging, including immunosenescence and inflamm-aging [15, 16]. Immunosenescence leads to significant shifts in the phenotypes and functionality of immune cells, with a reported decrease in naïve T cells and an increase in TEM and TEMRA cells in the old people [8]. Concurrently, inflamm-aging negatively impacts immunity by impairing the capacity of immune cells to respond to new antigen challenges, including vaccination ones [15].

The ISOLDA consortium was established and funded by the European Commission in 2020 to develop new strategies to address the poor efficiency of flu (and other) vaccines. This involves creating formulations with compounds that can mitigate the effects of the exacerbating inflamm-aging after vaccination. More specifically, at first, analyzing the immunophenotype of key subset cells involved in the vaccine response, we aimed to assess how the immune system, particularly T cell subpopulations, changes with age and in response to flu-vaccination. In this regard, some studies have reported a predominance of CD4 + naïve T cells in the immune response to vaccines, while memory CD8 + T cells are more prevalent in long-term defence [17]. Nonetheless, the literature on the effects of influenza vaccination on T cell populations is limited. At a second stage, we tested the effect of OLE and BIRB 796 as possible anti-inflammatory and antioxidant compounds able to reduce the inflammatory and oxidative stress induced by a viral stimulus in cultured T cells, reproducing the oxi-inflammatory stimulus generated by the administration of the vaccine. The combination of these two compounds has never been tested before, and their clinical application, particularly in the context of immune-related therapies such as vaccination, has not yet been developed.

Regarding the immunophenotype analysis, our results demonstrated statistically significant differences in CD8 + naïve T cells, TEM, and TEMRA percentages between the two analyzed age groups (Fig. 6b, f, and h). These findings are consistent with existing literature, describing the shifting distribution of these T cell subsets during the aging process [8]. The reduction in CD8 + naïve T cells with aging could impair the ability of the immune system in older individuals to mount an effective response after vaccination or any antigenic stimulation [18]. Specifically, comparing young to older adult groups, we have shown that CD8 + naïve T cells were significantly reduced, after vaccination, in the older adult group (Fig. 6b). TEM levels were higher at both T0 and T1 in the older population, with a statistically significant difference at T1 compared to the younger group (Fig. 6f). This suggests that the vaccine may play a role in shaping the immune response post-vaccination in older individuals, although our analysis does not confirm a direct change attributable to the vaccination itself because the same trend was observed at T0 although without statistical significance. Furthermore, no significant differences, within the same age group, at the different recruitment times (T0 and T1) were observed (Fig. 6). A distinct trend between T0 and T1 emerges exclusively for CD8 + TEMRA cells (Fig. 6h). In the younger group, these cells increase, whereas the old population exhibit a decrease over the same period (T0 vs. T1), although the difference is not statistically significant. Notably, TEMRA values consistently remain higher in the older adults compared to the young, reaffirming their association with age while suggesting an intriguing hypothesis about the potential effects of vaccination on this specific cell population. The increase in TEMRA cells with aging is closely associated with immunosenescence. However, the observed reduction of TEMRA cells after vaccination could suggest either a reactivation of the immune system or a reprogramming of the immune response toward an antigen-specific focus. Although not statistically significant, a rise in TCM and TEM cells, along with a decrease in naïve cells, was noted between T0 and T1, which appears to contradict the latter hypothesis. Additional experiments investigating the effects of vaccination on the distribution of memory T cells in the old population are needed to better clarify and define this pattern.

Also the analysis of the expression of exhaustion and senescence-like markers (i.e., CD28, and PD-1) could indicate a strongly impaired immune response to vaccination by inhibiting T cell activation and proliferation [13]. However, we did not obtain significant results for these cited markers, leading us unable to verify the contribution of specific immunosenescence factors in the vaccination response [18].

In older individuals, also the response to the vaccine and the duration of long-term protection are affected by the decline in antibody production [18]. In our study cohorts, the antibody response to all strains tested and included in the administered vaccine (Flucelvax® Tetra) remained higher than pre-vaccination levels at later time points for both analyzed populations (Figs. 4 and 5). Notably, in older adults, before vaccination, we observed a higher level of antibody titer against the H1N1 strain compared to younger individuals, although this difference was not statistically significant (Fig. 5a). However, at T1 and T2, respectively 21/28 and 56 days post-vaccination, in the older population there was a statistically significant decline in antibody levels compared to the younger group (Fig. 5b and c) although, within the older adults population group, there is an increase in antibody titer at these times compared to T0 (Fig. 4b) [13]. These data appear to align with findings in the literature about the efficacy of the tetravalent vaccine in eliciting humoral responses despite the age.

It should be emphasized that generally the antibodies titer towards virus strains observed in the older population (aged ≥ 65 years) was lower or comparable with seroconversion rates of younger groups, highlighting the dependence from age of vaccine’s effectiveness [19, 20]. Furthermore, the absence of a clear association between the cellular changes analyzed post-vaccination and antibody titer aligns with other studies that show that antibody titer does not correlate with the cellular response to the virus or vaccination [17, 21]. So, our data should confirm the lower response of older adults compared to the younger population in terms of antibody titers and consequently the probable lower vaccine efficacy linked to the humoral response [22].

Among the proposed strategies to enhance vaccination efficacy in the older adults, one of these is the mitigation of the inflammatory process, partly due to inflamm-aging, partly exacerbated by vaccination, targeting specific inflammation markers [23]. In this context, phenolic compounds from olive oil have demonstrated excellent anti-inflammatory and antioxidant properties in both in vitro and in vivo studies [9, 12]. The use of adjuvants derived from natural sources has become a key area of research, with particular focus on plant-based adjuvants such as saponins, tomatine, and others [24, 25]. These compounds show significant promise due to their dual role as immunopotentiators and anti-inflammatory. In addition, the use of plant-derived adjuvants offers enhanced safety profiles and potential environmental and economic benefits. Specifically, plant-derived adjuvants are highly valued for their biocompatibility, renewability, and ability to reduce reliance on synthetic chemicals, making them a crucial component in promoting sustainable practices [24, 25]. Specifically, in this work, it is hypothesized that OLE acts on the NF-κB pathway by modulating the production of pro-inflammatory (i.e., TNF-α, IL-6, and IFN-γ) and anti-inflammatory (i.e., IL-10) cytokines, as well as factors involved in the oxidative stress response [26, 27]. In particular, OLE has been shown to inhibit NF-κB activation by preventing the phosphorylation and degradation of its inhibitor, IκBα, thereby suppressing the nuclear translocation of NF-κB [11]. Additionally, its antioxidant effects may be linked to the interplay between NF-κB and other pathways, including those involving COX enzymes and Nrf2 [11]. By modulating Nrf2, oleuropein enhances the expression of antioxidant response elements, contributing to its protective effects against oxidative damage and inflammation [11]. Similarly, BIRB 796 has shown effectiveness as an inhibitor of p38 MAPKs, which are molecular targets for reducing inflamm-aging in adults [23, 27,28,29].

To study the effects on CD8+/CD4 + T cell populations producing key cytokines involved in the inflammatory process (TNF-α, IFN-γ, and IL-10), we tested OLE and BIRB 796, both individually and in combination, on the cultured PBMCs of voluntary donors at two recruitment times (T0 and T1), after the stimulation with a viral peptides stimulus from the H1N1 viral strain (PepTivator® Influenza A). Our investigations gave some explorative results about a possible role of OLE, alone or in combination with BIRB 796, in modulating CD8+/CD4 + TNF-α+/IFN-γ+/IL-10 + T cell levels, particularly in the older adults population, although without any statistical significance evidence (Figs. 7 and 8). The findings on the reduction of TNF-α + T cells by OLE, alone or in combination with BIRB 796, could be interesting due to the role of CD8 + TNF-α + T cells in inducing lung damage during flu infection and contributing to the increased production of pro-inflammatory cytokines and chemokines necessary for immune cell recruitment [17, 30]. Interestingly, the combination of OLE and BIRB 796 resulted in a probably more pronounced reduction in TNF-α levels than either BIRB 796 or OLE alone, suggesting a synergistic effect, attributed to BIRB and OLE’s inhibition of the p38 MAPK and NF-κB pathway respectively, which plays a key role in regulating inflammatory responses. This synergy could likely arise from the complementary mechanisms by which BIRB 796 and OLE impact molecular pathways involved in inflammation and oxidative stress, leading to a more effective suppression of TNF-α.

In the same way, IFN-γ is crucial in the antiviral and vaccination response. Its levels tend to decrease following vaccine administration and correlate with the antibody response in the general population [31]. However, in the older population, IFN-γ can have a negative role due to its ability to amplify the inflammatory process, which underlies the reduced vaccine response. In our analysis, OLE appears to probably act on IFN-γ-producing T cells, particularly in CD8 + T cells in the older adults group at T1 (Fig. 8d). OLE showed stable or slightly increased IFN-γ expression, reflecting OLE’s possible ability to support immune signalling pathways without excessive suppression of IFN-γ but behaving as a hormetic agent (Figs. 7d and 8c). This effect could be beneficial in the induction of virus elimination response during infections or vaccination itself by enhancing the immune response against viral antigens [32]. BIRB 796 did not seem to suppress IFN-γ, suggesting that it should not interfere with immune responses mediated by IFN-γ. However, when the compounds were combined, IFN-γ levels appeared to be maintained or modestly enhanced, which indicates a balanced immune response where inflammation is reduced, without compromising the antiviral and immune regulatory functions of IFN-γ.

Regarding IL-10 modulation, OLE displayed supposed minimal anti-inflammatory effects, as no significant increase in IL-10 + T cells was observed. In the older group, a slight reduction in CD8 + IL-10 + T cells was noted, particularly with OLE alone, suggesting a limited role in countering inflammation through IL-10 pathways (Fig. 8f). Interestingly, in the young group, OLE, in combination with BIRB 796, resulted in a decrease in CD4 + IL-10 + T cells (Fig. 7e), which may favor a pro-inflammatory immune profile more suitable for vaccine responses in this demographic area.

Also oxidative stress regulation involves both the NF-κB and p38 MAPK pathways, which can be activated by ROS/RNS, thereby promoting inflamm-aging. We have hypothesized that OLE and BIRB 796 could have reduced the effects of ROS and directly modulated their production [33,34,35]. Our analysis indicates that OLE, alone or in combination with BIRB 796, reduces ROS/RNS RFU values in both young and older adult populations (Fig. 9). However, we did not observe any effects on oxidative stress modulation related to the timing of recruitment. This suggests that the combined antioxidant effects of both agents may counteract oxidative stress, but we cannot make conclusions about their antioxidant effects concerning vaccination.

In summary, our exploratory findings suggest a role of OLE and BIRB 796 in the modulation of inflammation and immune responses, which is highly relevant for improved vaccine formulations. OLE could exhibit antioxidant and anti-inflammatory properties while preserving or slightly enhancing IFN-γ levels, a cytokine critical for antiviral immunity and effective vaccine responses. This suggests that OLE may act as a hormetic agent, reducing excessive inflammation while supporting immune signalling pathways that are essential for adaptive immunity. On the other hand, BIRB 796 should demonstrate an anti-inflammatory effect by targeting the p38 MAPK pathway, which is central to regulating inflammatory cytokines such as TNF-α. When combined, OLE and BIRB 796 could exert a synergistic effect, significantly reducing TNF-α levels while maintaining or modulating IFN-γ expression. This balance could be particularly crucial for vaccine formulations, where reducing inflammation can mitigate potential adverse effects, and preserving IFN-γ ensures robust activation of antiviral and immune-regulatory pathways.

These findings pave the way for the development of adjuvant systems that can modulate inflammation while preserving immune activation, offering a more tailored approach to vaccine development. However, it is important to acknowledge the several limitations inherent in this study.

The first of limitations observed in this study is the small sample size and, consequently, the low number of experiments conducted. The use of PepTivator® Influenza A H1N1 resulted in a poor in vitro response from T cells, which limited the number of events and constrained our ability to effectively apply the stimulus for cytokine-producing T cell determination. A similar observation has been reported by other researchers applying the same stimulus, where cytokine production was suboptimal [36,37,38,39]. The low rate of cytokine-producing T cells could also be attributed to the use of EDTA, used as anticoagulant in the collected blood samples, which may have decreased T-cell activation by disrupting calcium-dependent signalling pathways, thereby diminishing the cells’ capacity to produce cytokines [40]. The production of cytokines by T-cells may also have been influenced by the use of frozen cells. Although freezing and thawing procedures were performed properly according to standardized methods, and the cultured cells exhibited high viability, the freezing process may still have caused subtle functional impairments that could affect cytokine production. A more detailed analysis using tetramers and/or stimulating antigen-specific T cells with inactivated viral particles would increase the identification of antigen-specific T cells and thus the number of the same detectable [21, 31]. Additionally, we were unable to distinguish between cells activated by prior encounters with the viral antigen and those resulting from immunization with the influenza vaccine after the recruitment [21].

Despite the limitations encountered in our work, our preliminary results lay the foundation for future research on OLE and BIRB 796, both individually and in combination, in the modulation of the immune response, particularly in older adults and following antigenic stimulation. However, further investigations are needed to fully explore their interesting potential.