In addition to reduced mortality, heterologous beneficial BCG effects include decreases in infectious morbidities. Case control studies in Guinea-Bissau suggest that BCG vaccination and the presence of a scar among BCG-immunized infants was associated with a reduced risk of acute lower respiratory infection (ALRI) compared to unimmunized controls, with the association being stronger for females. In fact, children with ALRI were ~3-fold more likely to have not received BCG vaccine compared to children without ALRI. Similar results were found in an exploratory analysis of national health survey data from 33 low- and middle-income countries between 2000 and 2010, where 0-5 year-old BCG vaccinated children had 17-37% lower risk of suspected ALRI compared to unvaccinated controls. A retrospective epidemiologic study in Spain used data from the Official Spanish Registry of Hospitalizations to identify differences in hospitalization rates in BCG-vaccinated children (Basque Country, where universal neonatal BCG vaccination is practiced) as compared to non-BCG-vaccinated children (rest of Spain, where BCG is not routinely used). Analysis of 464,611 hospitalization episodes over a 15-year period showed that neonatal BCG immunization was associated with fewer hospitalizations for respiratory infections (the preventive fraction, defined as the attributable proportion of disease cases prevented by BCG exposure, was 40% and statistically significant among all age groups) and sepsis (preventive fraction 36%, statistically significant among the infant group). Differences diluted with age suggesting a time-limited protective effect of BCG vaccination vs. lower rates of hospitalization for respiratory infection in older children. No significant differences in the already low mortality rates were observed.
BCG scarring has been correlated with heterologous protective effects. A recent prospective study in rural Guinea-Bissau showed that children vaccinated with the BCG Moscow strain (also known as BCG Russia) who developed a scar had 26% lower mortality compared to children who did not develop a scar, mainly attributable to prevention of deaths from respiratory infections (mortality rate ratio [MRR] 0.2). This correlation of BCG scarring and improved survival has been replicated over different time periods and with different BCG strains; however, scarification rates differ by BCG formulation. For example, BCG Russia is less likely to produce a scar compared to BCG Japan and Denmark. BCG-induced scarring in Ugandan newborns was associated with higher IFNgamma responses to heterologous stimuli (tetanus toxoid, phytohaemagglutinin) at 1 year, and differed across strains (93% with BCG Denmark vs. 64% with BCG Bulgaria vs. 52% with BCG Russia). An RCT in Guinea-Bissau showed increased scarring induced by BCG Denmark and Japan compared to BCG Russia, but no significant differences in morbidity and mortality, at least by 6 weeks of age, possibly because BCG Russia also induced relatively high scarification rates in this cohort compared to others. Even though development of scarring also depends on additional factors such as vaccination technique, preservation of the cold chain, nutritional status of the recipient, age at time of vaccination and prior exposure to non-tuberculous mycobacteria, variable scarification rates may still predict variable heterologous protection in populations vaccinated with different BCG formulations.
In adults, BCG immunization induces specific epigenetic markers associated with the acquisition of a trained or tolerant phenotype after BCG vaccination. In healthy volunteers BCG induces trained immunity and heterologous protection from infections through epigenetic reprogramming of monocytes, specifically trimethylation of histone H3 at lysine 4 (H3K4me3) at the level of cytokine and TLR4 promoters. To further characterize BCG-induced innate immune regulation, adult PBMCs were cultured with BCG in vitro. Following heterologous stimulation with TLR ligands and bacteria, there was increased production of TNF, an effect mediated through the Nucleotide-Binding Oligomerization Domain Containing 2 pathway. In a randomized placebo-controlled adult study, yellow fever virus vaccine recipients who had been BCG vaccinated with the Denmark strain 1 month prior, had significantly lower yellow fever viremia compared to subjects who had received placebo vaccination. BCG vaccination conferred protection against yellow fever experimental infection by inducing genome-wide epigenetic reprogramming of monocytes involving genes related to signal transduction molecules, epidermal growth factor receptor, fibroblast growth factor, and vascular endothelial growth factor signaling pathways, as well as genes such as AKT1, MAPKs, and PI3K-related that have been shown to be important in beta-glucan-induced trained immunity, the prototypical trained immunity-inducing agonist in vitro. This effect correlated with induction of cytokine responses indicative of trained immunity: higher pro-inflammatory cytokine production (TNF, IL-1beta, IL-6) from BCG-vaccinated volunteers, compared to placebo-treated individuals, with a crucial role for IL-1beta production and release. These observations suggest potential mechanisms for heterologous protection that could also apply to infants, as epidemiological studies have shown that BCG vaccination results in lower all-cause mortality in infants.
More recently, immune-gene priming long non-coding RNAs (lncRNAs), positioned at the nexus of RNA, DNA, and protein interactions, have emerged as key regulators of gene transcription in trained immunity by positioning themselves at the nexus of RNA, DNA, and protein interactions. Taking advantage of the three-dimensional nuclear architecture and the close proximity of functionally related immune genes in topologically associated domains (TADs), lncRNAs contribute to accumulation of H3K4me3 at the promoters of trained immune genes in human monocytes.
Growing evidence that innate immune engagement by BCG enhances responses to other pathogens raises the possibility that some, or conceivably even most, of its clinical benefit is due to heterologous effects. However, the extent, mechanism and ontogeny of trained immunity in early life remain incompletely defined. Understanding how BCG-induced innate immune engagement, including BCG-induced enhancement of Th-polarizing cytokine production by antigen-presenting cells, varies by BCG strain and age is of basic and translational importance.
Intracellular metabolism plays key roles in regulating innate immune memory. In particular, different training programs induce metabolites that function as cofactors for epigenetic enzymes, which in turn induce chromatin and DNA modifications and modulate gene transcription upon re-challenge with a second stimulus. The Warburg Effect, first described in neoplastic cells, is a metabolic pathway important to trained immunity. Under normoxia, in resting cells, there is a low level of glycolysis and preferential pyruvate oxidation in the mitochondrion (oxidative phosphorylation), which confers slow but very efficient ATP production. In activated and proliferating cells, there is a metabolic switch from a state of oxidative phosphorylation to a state of glycolysis, crucial for the induction of the histone modifications and functional changes underlying BCG-induced trained immunity.
Epigenetic and metabolic reprogramming of hematopoietic progenitors may account for the long-term maintenance of trained immunity. Trained immunity affects myeloid cells as well as precursor cells of the innate immune system in the bone marrow. Administration of beta-glucan in mice induced selective expansion of myeloid stem and progenitor cells accompanied by a global increase in energy metabolism in bone marrow progenitors, particularly enhancement of cholesterol biosynthesis and glycolysis. Cytokine analysis in the bone marrow extracellular fluid revealed elevated IL-1beta levels, important in shaping immunometabolism within the bone marrow. In a randomized placebo-controlled human BCG immunization study with subsequent yellow fever vaccine challenge, reduction of viremia was highly correlated with the upregulation of IL-1beta, a cytokine associated with the induction of trained immunity, but not with the specific IFNgamma response, supporting a key role for IL-1beta as a mediator of trained immunity responses. In mice, access of BCG to the bone marrow reshaped the transcriptional landscape of hematopoietic stem cells resulting in preferential myelopoiesis vs. lymphopoiesis and generation of macrophages that provided improved protection against TB.
Changes in glucose, glutamine and cholesterol metabolism enable maintenance and longevity of trained immunity via accumulation of immunologically active intermediate metabolites. Examples include: (a) cholesterol, which participates in cell membrane remodeling and increased sensitivity to subsequent stimuli, (b) succinate and fumarate, which antagonize histone demethylation and suppress anti-inflammatory genes, (c) acetyl-CoA, an essential substrate for acetylating processes, and (d) NAD+ which is important for epigenetic changes resulting in a switch from glucose to fatty acid oxidation during LPS-induced tolerance and sepsis-induced immune paralysis. Immunometabolic changes may be different between newborns and adults, reflecting the differential nutritional and metabolic needs of the two groups, as well as their distinct immune response to pathogens. Indeed the ontogeny of immunometabolism is an emerging and promising area of research (see Box 2).
Systems vaccinology, the application of global molecular techniques such as metabolomics, proteomics, or transcriptomics, can provide unique insights into vaccine-induced immune responses by identifying molecular signatures that may predict and give insight into vaccine-induced immunogenicity and protection. The systems biology can provide valuable insights into host-pathogen interaction with Mtb as well as generate tools for early and proper diagnosis of TB, identification of BCG protective efficacy, and accelerated development of better TB vaccines. The metabolome, the inventory of all metabolites present in a given sample, reflecting both genetic and epigenetic influences, shifts upon immune activation and can in turn shape immune responses. Metabolic phenotype influences vaccine immunogenicity and together with orthogonal datasets can identify correlates of vaccine immunity. Lipid metabolism is pivotal in the regulation of inflammatory signaling hence making lipidomics, an in-depth profiling of lipid metabolites, a valuable modality as well. Lipid metabolism regulates immune cells via cell membrane synthesis and is important to epigenetic reprogramming of immune cells. Mass-spectrometry-based metabolomics, together with computational tools, can identify and correlate metabolic pathways between samples, providing a powerful approach for clinical diagnostics. More studies are warranted to build the area of biomarker identification while addressing the challenges of identifying correlates of protection against TB.
BCG has been recognized as a potent immunomodulator for decades with extensive use for cancer and particularly bladder cancer treatment. In the past decade, there has been revived interest in BCG vaccine for potential new therapeutic uses in type 1 diabetes mellitus and treatment of other forms of autoimmunity. When administered to young NOD (autoimmune-prone) mice, BCG could not only stop new-onset diabetes but also reverse end-stage diabetes, owing to induction of suppressive regulatory T cell (Treg) expansion, thereby preventing the immune system from attacking the body's own tissue. In a clinical trial involving humans with longstanding type 1 diabetes mellitus, repeat BCG administration (2 doses) led to transient restoration of pancreatic cell islet function in vivo (for 4-6 weeks after vaccination). The suspected mechanism was BCG-induced proliferation of Tregs and selective elimination/suppression of auto-reactive cytotoxic T cells, possibly via TNF induction/TNF receptor 2 agonism. Long-lasting improvements in glycemic control as evidenced by sustained decreases in hemoglobin A1c were achieved via accelerated glucose utilization induced by a systemic shift from oxidative phosphorylation to aerobic glycolysis.
In a double-blind, placebo-controlled trial conducted in Italy involving subjects with early symptoms consistent with multiple sclerosis (MS), participants were randomly assigned to receive BCG or placebo and monitored monthly with brain Magnetic Resonance Imaging (MRI) (6 scans). By the end of the study, 58% of those vaccinated had not developed MS, compared with 30% of those who received placebo. Overall clinical benefits after BCG administration in new onset MS were durable and even enhanced at 5 years. In another trial, BCG vaccination was found to decrease MS disease activity and prevent progression of brain lesions in patients with relapsing-remitting MS. A phase III clinical trial of BCG to reverse progression of MS is now underway.
BCG vaccination has also been associated with a reduced risk of atopic disorders as noted in a Japanese cohort, as well as in African children, where the reduction in atopy associated with BCG was greater the earlier the age at vaccination, with the largest reduction seen in children vaccinated in the first week of life. This observation is consistent with BCG being a powerful inducer of a Th1 phenotype in infants and shifting their immune response away from the Th2-type that is typically favored in early life. Importantly, these immune polarizing effects of BCG may be yet another result of trained immunity, which may contribute to host survival in early life and affect the risks of infection, allergic and chronic inflammation later in life. A randomized controlled trial to determine if BCG immunization at birth reduces allergy and infection in infants is currently underway in Australia (Melbourne Infant Study, NCT01906853).
Few studies have investigated the influence of age at and timing of immunization on BCG-induced immunogenicity and protection against TB. BCG-specific effector CD4 T cell responses demonstrate increased antigen-specific CD4 T cell proliferative capacity in infants compared to older children. Vaccination at birth induces a broad Th1/Th2/IL-17/Treg anti-mycobacterial response but the Th1/Th17 response is reduced when delaying the vaccine from birth to 4 1/2 months of age. In a randomized trial of low birth weight newborns, BCG significantly increased in vitro whole blood cytokine responses to heterologous TLR agonists and to PPD in infants 4 weeks post-vaccination, particularly cytokines IL1beta, IL-6, TNF, and IFNgamma, potentially contributing to broad protection against infections. These studies illustrate that timing of BCG administration can be crucial for its immunogenicity with distinct effects depending on which outcomes are studied (mycobacterial-specific vs. heterologous). Mechanistic studies are needed to provide a basis for understanding the impact of immune ontogeny on BCG immunogenicity.
Comparable CD4 and CD8 T cell anti-mycobacterial responses and whole blood cytokine production were noted in Australian infants who received BCG Denmark at birth (early BCG) compared to 2 months after birth (late BCG). However, in TB-endemic regions such as Cape Town and South Africa, delaying immunization with BCG Denmark 10 weeks post-birth led to increased frequencies of memory CD4 T cells at 1 year of age. These two seemingly contradictory studies emphasize the importance of immune ontogeny, as well as genetic and epigenetic host factors, including prior and ongoing host exposure to non-tuberculous mycobacteria, to the immunogenicity of live vaccines.
BCG vaccination in children results in different cytokine induction patterns compared to adults. Vaccine efficacy rates were indeed higher in studies conducted in populations vaccinated during childhood compared with populations vaccinated at older ages. The longevity of BCG clinical effects remains largely unknown and may in part depend on age of immunization. In the largest community-based controlled trial of BCG vaccination conducted in southern India in the 1960s, vaccine recipients were reevaluated 15 years after BCG vaccination: protective efficacy in persons who had been vaccinated as children was found to be 17%, while no protective effect was seen in people who had been vaccinated as adolescents or adults.
WHO currently recommends BCG at birth for countries where TB is endemic since birth is the first point of contact for the newborn with the healthcare system. In practice, however, many healthcare systems continue to institute policies such that BCG is not administered unless a certain number of infants are present to receive immunization from the multi-dose BCG vial resulting in missed opportunities to administer it at the earliest possible age per WHO recommendations. However, based on the above, a "one size fits all" policy on optimal BCG timing may not be realistic and immunization should be tailored to different global populations with different risk factors in different settings. Further investigations involving the ontogenetic aspects of BCG-induced immunogenicity and protection against TB are needed. Highly standardized comparison studies should account for the environmental (local and regional) exposure, genetic and epigenetic factors, biological age, and immunological status of vaccinated participants. Such studies would further inform the variation of heterologous effects seen as a result of BCG vaccination.
The ability of live vaccines such as BCG to induce heterologous immunity raises the possibility of leveraging such broadly protective effects in the development of novel vaccine formulations, in the form of "trained immunity-based" vaccines. Firstly, increased awareness of innate memory may be employed to define new classes of vaccine adjuvants, crucial tools to optimize current vaccines and develop new ones. Adjuvants enhance responses to vaccine antigens by a variety of mechanisms, but like BCG, many are capable of acting via PRR signaling (e.g., TLRA), which possibly could hold the potential of inducing innate memory and could thereby mediate long-term changes in host defense. Also, recent advances in adjuvant discovery and delivery have opened up a new toolbox on how vaccinologists can employ adjuvants, including synthetic small molecule PRR agonists. Thus, to confer protective immunity a strategy might be the combination of adjuvants, with potential of inducing beneficial non-specific trained immunity responses, formulated along with the specific selected antigen epitopes. An important aspect to take into account is that it is not yet known whether or not all PRR stimuli produce trained immunity-like responses. As different adjuvants may trigger different cell activation pathways and have age-specific activity, it is likely that more than one trained immunity pathway could be targeted for perturbation. In addition, putative target cell populations for innate training may vary, including progenitor cells, tissue resident or circulating monocytes, which may be optimally targeted via specific routes of administration or by rationally selected adjuvant formulations.
Secondly, characterizing mechanisms by which BCG enhances neonatal immunity may inform rational design of scalable, synthetic subunit vaccine formulations for newborns. Initially, TLR7/8a imidazoquinolines were shown to induce trained immunity in newborn mice, raising the possibility that such an approach could generate a vaccine that may also induce "BCG-like" trained immunity. However, since free un-formulated molecules may have off target effects, another approach is to build "BCG-like" synthetic "non-live" particulate vaccines that may mimic BCG's immune-enhancing effects. Inclusion of an imidazoquinoline small molecule TLR8 agonist in a polymersome nanoparticle (~150 nm diameter) induced robust Th1 polarizing responses from human newborn monocyte-derived dendritic cells in vitro that at least matched and for some biomarkers such as IL-12p70 exceeded those induced by BCG Denmark. Of note, when co-loaded with the M. tuberculosis antigen 85B peptide 25, the TLR8-agonist containing polymersome nanoparticles were comparable to BCG in inducing antigen-specific T cell responses in human TLR8-expressing neonatal mice in vivo. This is promising, since BCG reduces the risk of disseminated early life TB safely, elicits Th1-type neonatal immune responses and requires only a single dose at or shortly after the time of birth. The key role of TLR8 agonists for protection against Mtb challenge was recently verified by others with humanized TLR8 mice and in human studies, wherein humans with hypermorphic alleles of TLR8 demonstrated enhanced BCG-induced protection against TB.
Thirdly, the robust safety and immunogenicity profile of BCG has rendered it an attractive vector for vaccine development against other infectious diseases. Recombinant BCG technology has been studied in the context of vaccination against HIV, Lyme disease, malaria, measles, and HCV. When administered in early life, BCG can act as an adjuvant enhancing antibody responses to recombinant hepatitis B surface antigen (rHBsAg) both in mice and in human infants. In another approach, a recombinant strain of M. bovis BCG that secretes high levels of functional murine monocyte chemotactic protein 3 (BCGMCP-3) attenuated vaccine virulence in immunodeficient mice, while maintaining protective efficacy against Mtb in mice by enhancing antigen-specific IFNgamma T cell responses, as compared to a control BCG (Pasteur strain 1173P2). A recombinant BCG strain expressing listeriolysin O to enhance cytosolic entry of BCG antigens for MHC I presentation, named VPM1002, induced both CD4 and CD8 responses and demonstrated safety and immunogenicity in a phase 2 clinical study in South African newborns. Overall, insights into BCG vaccine-induced heterologous and specific immunity may provide insights into the development of a broad spectrum of anti-infective vaccine formulations.