Immunomodulatory Effect of the Bacillus Calmette–Guérin (BCG) Vaccine on the In Vitro Interferon Response Induced by Respiratory Syncytial Virus (RSV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antigens

The bacillus Calmette–Guérin (BCG) vaccine, which contains a live, attenuated strain of Mycobacterium bovis, has been a cornerstone of tuberculosis prevention for decades. Despite its wide use, much remains unknown about its mechanisms of action. Historically, BCG was thought to induce primarily a heterologous immune response involving memory T and B cells, which develop over several weeks and do not contribute to immediate perinatal immunity (Biering-Sørensen et al. 2012). However, recent research proposes an additional mechanism called “trained immunity,” in which innate immune cells such as macrophages, natural killer cells, dendritic cells (DCs), and hematopoietic stem cells in the bone marrow are trained (Netea et al. 2011). This training enables these cells to perform memory-like behavior, potentially explaining the short-term actions of immune cells that gain long-term memory capacity (Kaufmann et al. 2018; Mitroulis et al. 2018; Liu et al. 2024b).

Epigenetic modifications, in particular histone modifications, play a key role in the process of trained immunity (Sviridov et al. 2022). After BCG vaccination, an increase in H3K4me3 methylation is observed in the promoters of key pro-inflammatory cytokine genes, increasing their activation (Kleinnijenhuis et al. 2012; Arts et al. 2015). The vaccine also affects cellular metabolic pathways, including glycolysis, oxidative phosphorylation, and glutamine catabolism, which are essential for the growth and function of trained immune cells. This metabolic shift toward glycolysis further promotes histone modification and BCG-induced trained immunity.

The benefits of the BCG vaccine extend beyond tuberculosis, as evidenced by its protective effect against a variety of nonspecific infections, thereby reducing morbidity and mortality, especially in children (Uthayakumar et al. 2018). The vaccine is effective against bacterial and viral pathogens, including herpes virus, influenza virus, and respiratory syncytial virus (RSV) (Starr et al. 1976; Stensballe et al. 2005; Mukherjee et al. 2017). Studies have shown that BCG vaccination modifies the effectiveness of antiviral vaccines and improves the immune response to viral infections. Recent studies are even exploring the potential of BCG in the development of new vaccines, such as the recombinant BCG vaccine against RSV, which has shown promising results in animal models (Arts et al. 2016). Kaufmann et al. (2022) observed a reduction in morbidity and mortality due to influenza A virus in a mouse model administered the BCG vaccine. Moreover, monocytes from BCG-vaccinated people showed an increased cytokine response. It is worth noting that BCG has been shown to not only enhance the body’s response to viral infections but also increase the effectiveness of antiviral vaccines. For example, a study in a group of healthy volunteers showed that BCG vaccination enhances the effect of the influenza vaccine by increasing the production and persistence of interferon (IFN)-γ (Leentjens et al. 2015). Studies conducted in Guinea-Bissau also indicate that young children who received the BCG vaccine had a lower incidence of acute respiratory infections caused by RSV (Stensballe et al. 2005). Additionally, current research efforts are focused on developing a vaccine against RSV using the BCG strain. In an animal study in newborn calves, a recombinant BCG vaccine expressing hRSV nucleoprotein (rBCG-N-hRSV) was shown to induce both humoral and cellular responses against RSV (Díaz et al. 2021). Additionally, a study on adult volunteers confirmed the safety, protective properties, and increased production of interleukin-2 and IFN-γ of this vaccine (Abarca et al. 2020).

In viral infections, the innate immune response is the main line of defense, and IFNs are the key cytokines produced. IFNs help block virus binding to host cells, inhibit nucleocapsid release from the viral envelope, limit viral messenger RNA (mRNA) translation and viral protein synthesis, and promote immune cell activation and recruitment (Kaur and Secord 2021; Liu et al. 2024a). Type 1 IFN (including IFN-α and IFN-β) and type 3 IFN (IFN-λ) are particularly associated with the antiviral response (Chong et al. 2022; Ogger et al. 2022). The role of type 2 interferon (IFN-γ) during viral infections such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has not yet been fully elucidated. Although IFN-γ, IFN-α, and IFN-β, are critical in combating RSV infection, research indicates that type I IFNs may play a limited role in responding to RSV infection (Blasius and Beutler 2010). Furthermore, Marr et al. (2014) observed an increase in IFN-α production in RSV-infected primary plasmacytoid DCs, which becomes more pronounced with age.

In light of these findings, our study examined the effect of the BCG vaccine on the IFN response to SARS-CoV-2 and RSV antigens in vitro, to understand how BCG may enhance immune defense against these viruses. Our study aimed to investigate whether the BCG vaccine modulates the antiviral response of blood cells from BCG-vaccinated individuals upon co-stimulation with viral agents. While vaccination with BCG vaccine during viral infection is not the standard practice, we aimed to investigate the potential for the long-term immunological effects of BCG vaccine. Considering that early exposure to the pathogen may influence the intensity of the immune response, our study examined in detail the effect of BCG mycobacteria on IFN-α, IFN-β, and IFN-γ mRNA expression and protein secretion in whole blood cultures from BCG-vaccinated seropositive seronegative RSV/SARS-CoV-2 Polish children. An additional aim of our study was to elucidate the potential modulating role of early viral exposure on the BCG-induced IFN response. The in vitro model used in our study represents a controlled method to evaluate the effects of the BCG vaccine in combination with viral antigens. Although this approach does not fully replicate the physiological conditions, it provides a valuable framework for analyzing the immunological and molecular mechanisms. This model enables a detailed exploration of vaccine interactions and immune responses, offering insights that might not be feasible in vivo. Understanding these mechanisms has the potential to inform clinical strategies, particularly in optimizing vaccine design and combination therapies.

To elucidate the role of the BCG vaccine in modulating the immune response to viral infections, it is important to examine the multifaceted aspects of immune system activation, training, and response modulation that BCG offers. Trained immunity through BCG involves epigenetic and metabolic reprogramming of innate immune cells, especially monocytes and macrophages, resulting in increased and more rapid production of pro-inflammatory cytokines during subsequent infections. These long-lasting effects may provide nonspecific protection against a variety of pathogens beyond Mycobacterium tuberculosis (Moorlag et al. 2019; Faustman et al. 2022). Furthermore, research suggests that BCG may alter the bone marrow microenvironment, enhancing the functionality of hematopoietic stem cells and progenitors, and thereby expanding the scope of immune defense (Arts et al. 2018).

In particular, our research focused on the effect of the BCG vaccine on the production of IFNs, which play a key role in the antiviral response. Type I interferon, which includes IFN-α, is known to be a key element involved in virus clearance and regulation of the host immune response. Several studies have shown that a strong IFN-α response is associated with protection and a milder course of RSV infection in infants (Hijano et al. 2019). Unfortunately, it is known that the innate immune response, especially the production of type I interferons, is suppressed by RSV in infants, which can lead to serious disease progression (Ramaswamy et al. 2006). In our study, stimulation with RSV peptides alone resulted in detectable levels of IFN-α, which were higher than those observed in unstimulated cultures. However, the magnitude of this induction was relatively low compared with other stimulation conditions, suggesting that RSV peptides alone have a limited capacity to induce IFN-α production. This may indicate that additional signals or co-stimulated cultures with other immune activators are necessary to elicit a more robust IFN-α response. These findings underscore the complexity of IFN induction pathways and highlight that RSV peptides alone may not fully engage the immune mechanisms required for strong type I interferon production. Upon assessing the effect of BCG mycobacteria on the production of type I interferon (IFN-α) in response to RSV and SARS-CoV-2 virus antigens, significant discrepancies were observed in culture supernatants. It was found that the administration of live BCG bacilli increases the production of IFN-α in response to RSV antigens in all groups except the uninfected cohort. A particularly marked difference in the production of IFN-α was observed in blood cultures of children from the SARS-CoV-2(+) and SARS-CoV-2(−) RSV(−) groups. This indicates that the BCG vaccine can specifically modulate the immune response. The literature suggests that RSV can hinder signaling of the IFN-α synthesis, potentially reducing its production (Ramaswamy et al. 2006). Conversely, when cultures were performed in the presence of SARS-CoV-2 virus antigens, live BCG mycobacteria did not induce an increase in IFN-α levels compared with cultures stimulated with antigens alone. While BCG triggers immune responses to various pathogens, our data on IFN-β and IFN-γ indicate that these mechanisms are not distinctly different for each pathogen.

Interferons play a crucial role in defending against SARS-CoV-2. It has been shown that IFN-α can inhibit viral replication, but SARS-CoV-2 may suppress IFN-α signaling, and genetic variations in the IFNRA2 gene are linked to disease severity (Vanderheiden et al. 2020; Bessière et al. 2021; Akter et al. 2022; Znaidia et al. 2022). However, SARS-CoV-2 has developed strategies to evade host responses by suppressing type I interferon signaling (Beyer and Forero 2022). For example, angiotensin-converting enzyme 2, the entry receptor of SARS-CoV-2, can interfere with the IFN-α signaling pathway, inhibiting its production and thus facilitating viral infection (Chen et al. 2022). Although the benefits of prophylactic administration of IFN-α in the early stages of the disease are clear, questions remain regarding the feasibility of using IFN-α in patients with severe COVID-19. Despite initial reports indicating decreased IFN production, emerging evidence suggests that people with severe COVID-19 have a sustained response to type I IFN. This is in contrast to the delayed and potentially suppressed response to IFN observed in the early stages of infection. The study found no significant differences in IFN-α levels between cultures stimulated with SARS-CoV-2 antigens alone and cultures stimulated with live BCG bacilli and SARS-CoV-2 antigens. However, in the group seropositive for RSV, the expression of IFNA5 mRNA was significantly higher in cultures stimulated with live BCG bacilli and SARS-CoV-2 antigens compared with cultures stimulated with SARS-CoV-2 antigens alone. The lack of differences in IFN-α secretion may suggest post-transcriptional mechanisms regulating the secretion or translation of this IFN. This phenomenon may explain the observed blocking of the secretion of larger amounts of IFN-α protein, despite higher expression of IFN-α mRNA. Additionally, the immune system may have regulatory mechanisms that limit excessive production of IFN-α despite elevated mRNA levels to prevent an excessive inflammatory response that may be harmful. The results indicate that stimulation of IFN-α gene transcription by live BCG mycobacteria in combination with SARS-CoV-2 antigens is due to higher mRNA expression. However, alternative mechanisms at the post-transcriptional level may impede both the production and secretion of the IFN-α protein. It is noteworthy that the significant increase in mRNA levels for the three IFNs tested after stimulation with BCG and RSV antigens provides insight into transcriptional activation of immune pathways. The discrepancy between mRNA levels and protein production highlights a potential role for post-transcriptional regulation, such as mRNA stability, translation efficiency, or protein degradation. Further analysis of post-transcriptional mechanisms and secretion studies may help to clarify these issues.

Previous studies have shown that IFN-β is a key component of the immune response to RSV infection, modulating immunity and inhibiting viral replication (Antunes et al. 2019; Hijano et al. 2019). Notably, Lo et al. (2005) demonstrated that RSV develops mechanisms to attenuate the host response to IFN-β. RSV unstructured proteins (NS1 and NS2) have been observed to downregulate Stat2, a key player in the IFN-α/β signaling pathway, thereby interfering with the IFN response and facilitating host evasion. The results of our study support this hypothesis, as IFN-β secretion levels in our experimental cultures were low and did not differ significantly from those in the control groups. This indicates that the stimulation conditions used in our experiments may not have been sufficient to elicit a robust IFN-β response or that variability among replicates may have obscured any potential differences. These findings suggest that additional factors or alternative experimental approaches might be required to better characterize IFN-β production under these conditions. Moreover, in the context of SARS-CoV-2 infection, IFN-β plays a pivotal role in the host immune response. A study by Li et al. (2020) showed that the ORFF6 and ORF8 proteins, as well as the nucleocapsid proteins of the SARS-CoV-2 virus, can inhibit the IFN-β signaling pathways, thereby enhancing the infection. In the present study, no significant differences were observed in the levels of IFN-β secretion and expression across the experimental conditions. This could suggest the involvement of regulatory mechanisms that limit IFN-β production, such as feedback inhibition by suppressor of cytokine signaling proteins or the action of regulatory cytokines, which are known to modulate IFN responses. Alternatively, it is possible that the stimulation conditions used, including the duration or type of stimuli, were not sufficient to robustly induce IFN-β expression, allowing such inhibitory mechanisms to dominate. These findings highlight the complexity of IFN-β regulation and suggest that further investigation is needed to elucidate the specific factors influencing its production in this context.

The BCG vaccine has gained attention for its potential role in enhancing antiviral immunity, particularly through modulating the IFN-γ response (Weir et al. 2006; Lalor et al. 2010, 2011; Corral-Fernández et al. 2016). Our study showed that BCG increased the production of IFN-γ in co-stimulated cultures with RSV or SARS-CoV-2 antigens. This aligns with previous research indicating that BCG boosts IFN-γ production even against non-mycobacterial infections (Kleinnijenhuis et al. 2012, 2014). IFN-γ stimulates antigen presentation by inducing the expression of major histocompatibility complex (MHC) molecules and promotes the cytotoxic activity of virus-specific Natural killer (NK) cells and T cells. The early IFN-γ response plays a key role in influencing the course of viral infection. Consequently, decreased production of IFN-γ during RSV infection may serve as a determinant of disease severity. Bont et al. (2001) showed that the severe course of the RSV virus is associated with reduced secretion of IFN-γ in the blood. Furthermore, Aberle et al. (1999) observed a reduction in IFN mRNA expression in the context of RSV-induced bronchiolitis. Our study also demonstrated the lack of strong IFN-γ secretion and IFN-γ mRNA expression in whole blood cultures stimulated with isolated RSV antigens. However, in co-stimulated cultures, live BCG mycobacteria significantly increased IFN-γ production in response to RSV antigens, which may indicate that the BCG vaccine may specifically modulate the immune response and provide protection against RSV. It is worth noting the potential interaction between prior RSV infection and the immunomodulatory effects of BCG vaccination. One possible explanation is that RSV, as a respiratory virus with distinct immunological signatures, may elicit a more robust activation of trained immunity pathways when combined with BCG-induced immune modulation. This is supported by the observed increases in interferon production, particularly IFN-γ, which is known to play a key role in antiviral defenses. The study by Eichinger et al. (2015) in an animal model provides evidence that treatment with IFN-γ protects against RSV infection. This shows that IFN-γ is of great importance in reducing viral load and preventing serious disease progression.

BCG has also been proposed to prevent SARS-CoV-2 infection due to its immunostimulatory properties. Studies show that BCG enhances IFN secretion, potentially benefiting COVID-19 patients (Nguyen et al. 2021; van Laarhoven et al. 2021). Hilligan et al. observed that intravenous BCG vaccination in mice leads to a sustained strong IFN-γ response in the lungs, which may promote early control of SARS-CoV-2 replication (Hilligan et al. 2022, 2023). Similarly, our study found higher IFN-γ levels in BCG + SARS-CoV-2 co-stimulated cultures, indicating that BCG stimulates T cells to produce IFN-γ, counteracting SARS-CoV-2’s suppression of IFN responses. Differences in IFN-γ levels between RSV/SARS-CoV-2 seropositive and RSV seropositive groups suggest complex interactions in co-infections. The observed differences are consistent across independent experimental replicates, suggesting a reproducible pattern rather than random variation. Small changes in the levels of cytokines, such as IFN-γ, can have a disproportionately large effect on the immune response. Experimental conditions designed to simulate viral infection scenarios can amplify subtle changes in cytokine expression, highlighting a context-dependent significance that requires further study. It is conceivable that an increased IFN response may have a beneficial effect on the response to viral infections, but it is important to remember that an excessive pro-inflammatory response, including the production of type I IFN, can be detrimental, causing a cytokine storm. Our findings suggest that co-infection with RSV and SARS-CoV-2 affects the BCG-induced immune response, particularly IFN-γ production. We observed lower IFN-γ levels in the RSV/SARS-CoV-2 co-infected group compared with the RSV-only group. One possible explanation for this observation is immune interference between the two viruses. SARS-CoV-2 may alter the immune response to RSV, potentially through immune exhaustion, which can impair the ability to respond effectively to both pathogens. Alternatively, BCG-induced responses may redirect focus in the presence of dual infections, reducing IFN-γ production for RSV. These mechanisms warrant further investigation, especially for vaccine-based therapies like BCG in the context of co-infections.

Our results showed significantly higher serum levels of IFN-α and IFN-γ in the SARS-CoV-2 seropositive group, suggesting a preactivated immune state. The elevated levels of IFN-α and IFN-γ in the serum of SARS-CoV-2(+) individuals likely reflect the heightened immune activation associated with the infection. IFN-α is a type I interferon known to play a key role in the antiviral response by promoting viral clearance and activating innate immune cells, while IFN-γ, a type II interferon, is primarily produced by activated T cells and NK cells to enhance adaptive immunity and macrophage activation. The observed increase in these cytokines may be attributed to the host’s attempt to control viral replication and modulate the immune response during infection. However, persistent elevation of these cytokines has also been associated with hyperinflammatory states and disease severity in COVID-19. This dual role highlights the complexity of IFN responses in SARS-CoV-2 infection. Differences in the immune response to RSV and SARS-CoV-2 have been observed in different age groups. Children generally show a stronger immune response to SARS-CoV-2 than adults. This may be influenced by the initial immune training following BCG vaccination, which is common in many countries for children. The distinct pathophysiological characteristics of these viruses, with RSV having a more pronounced impact on the lower respiratory tract in young children than SARS-CoV-2, may be influenced in part by the trained immune profile established by BCG vaccination early in life. The results of our study indicate that the BCG vaccine may have an immunomodulatory effect on the IFN response induced by SARS-CoV-2 and RSV viruses. The different magnitudes of the observed responses may indicate the existence of separate immunological mechanisms stimulated by BCG in response to different pathogens. Further research on broad population groups is necessary to clarify the molecular basis of the observed phenomenon and its potential clinical significance, especially in the context of using the BCG vaccine as a regulator of the immune response to viral infections. Our in vitro model provides a controlled environment to evaluate the effects of the BCG vaccine in combination with viral antigens. While it does not entirely mimic actual physiological conditions, it enables a focused analysis of molecular and immunological mechanisms underlying vaccine–antigen interactions. These insights could have significant implications for clinicians, particularly in designing effective immunotherapeutic strategies and understanding immune modulation induced by such vaccine combinations. By delineating the immune pathways activated in this controlled setting, the study lays the groundwork for translating these findings into clinically relevant applications. Our study does not recommend BCG vaccination during active viral infection, but rather investigates the immunomodulatory potential of BCG-trained immunity in vitro as a proof-of-concept.

The observed uniformity in cytokine production patterns across seropositive and seronegative individuals suggests that in our in vitro experimental settings, cytokine responses were more dependent on the stimuli used than on immune memory or prior exposure history. This result aligns with the hypothesis that innate immune responses, such as those triggered in vitro, may overshadow or mask the contributions of adaptive immune memory in this context. The controlled environment of our experiments and the specific nature of the stimuli (RSV or SARS-CoV-2 antigens) may not have been sufficient to differentiate the nuanced memory responses that might occur in vivo. Additionally, innate immune responses, such as those associated with trained immunity or cross-reactivity, may have overshadowed adaptive memory effects in our assays. It is reasonable to hypothesize that individuals with prior exposure (seropositive) might exhibit either a quantitative or qualitative difference in cytokine response compared with seronegative individuals due to immune memory. One might expect higher cytokine production in RSV(+) individuals, particularly in responses involving memory T cells or other adaptive immune components. However, it is also possible that memory-driven responses would differ in their cytokine profile rather than magnitude, depending on the nature and strength of the prior immune encounter. Similarly, we might anticipate heightened cytokine responses in SARS-CoV-2(+) individuals, reflecting the presence of adaptive immune memory. However, the magnitude and profile of the response could vary based on the time elapsed since infection, the antigen used in the assay, and the individual’s immune status.

The exclusion of unvaccinated children in our study is a significant limitation that hinders the generalization of our findings to the broader population. Our conclusions are focused solely on the immune mechanisms in BCG-vaccinated children, which may not reflect the immune responses in the general population. Due to the retrospective nature of data collection, information on the sequence of infections was not available for all participants, highlighting the need for further prospective studies that account for these variables. Further research is warranted to explore how the immunomodulatory properties of BCG can be optimized for current and emerging viral threats. To fully realize the potential of the BCG vaccine in modulating immunity against viruses, it is necessary to consider both its proven benefits and the challenges associated with its wider use. The vaccine’s affordability and existing supply chains make it a valuable tool in low-resource settings, potentially offering a cost-effective strategy to enhance global health security against viral epidemics. However, variability in immune responses due to genetic differences between populations poses a significant challenge, highlighting the need for targeted research to enhance BCG’s effectiveness in different demographic groups. Ongoing global trials are examining the effectiveness of BCG in preventing infections other than tuberculosis (Kleinnijenhuis et al. 2014). It is worth noting that we only included one time-point in our study, 48 h after stimulation, which is a limitation in terms of fully understanding the dynamics of cytokine expression. It is known that there is a difference in the kinetics of mRNA and protein expression of cytokines, with mRNA expression often peaking much earlier than protein levels. Therefore, while a time-point of 48 h may be appropriate to assess the peak of cytokine expression at the protein level, it may be too late to capture changes at the mRNA level that may occur earlier. Furthermore, mRNA analysis allows measurement of the transcript at one specific time-point, which may not reflect the full dynamics of the transcription process. In contrast, measuring protein in the supernatant provides information on cytokine accumulation or consumption at a specific time-point, which may be more optimal for the long-term cellular response. These studies are of great importance in determining the role of the vaccine in the broader context of treating infectious diseases. They may also guide future vaccination strategies to include BCG as a permanent part of vaccination schedules around the world, especially in regions most vulnerable to emerging pathogens. It is critical to remove potential barriers, including regulatory approvals and public perception, to facilitate the successful repositioning of BCG. Additionally, public health communication must evolve to support the understanding of new and potential uses of BCG. This is essential to ensure that communities are informed and open to revised vaccination protocols.

In summary, while the BCG vaccine shows promise in enhancing the immune response to a range of viral infections, fully realizing this potential will require a coordinated approach across research, health policy, and community engagement. Results from ongoing research will be critical in shaping the future of infectious disease prevention and management by using an old vaccine to address new public health challenges.

The conducted studies indicate that the BCG vaccine may have an immunomodulatory effect on the IFN response induced by SARS-CoV-2 and RSV infections. Differences in the intensity of this response may suggest the existence of distinct immune mechanisms stimulated by BCG in reaction to various pathogens. The observed variability in the immune response highlights the complexity of BCG’s action and underscores the need for further comprehensive investigations. Additional research on large and diverse population groups is necessary to elucidate the molecular basis of the observed phenomenon and its potential clinical significance. Such studies should identify specific biomarkers and genetic factors that contribute to the differential immune responses. Understanding these mechanisms is crucial, especially in the context of utilizing the BCG vaccine as a potential regulator of the immune response to a broad spectrum of viral infections. Moreover, these insights could pave the way for new therapeutic strategies and improve the efficacy of existing vaccines against emerging viral threats.