Is There a Role for Immunostimulant Bacterial Lysates in the Management of Respiratory Tract Infection?

Abstract

Bacterial Lysates are immunostimulants clinically prescribed for the prevention of respiratory tract infections (RTIs). It has been shown that Bacterial Lysates upregulate the immune system, acting both on innate and adaptive reactions. In fact, there are demonstrations of their efficacy in restoring the integrity and immune function of epithelial barriers, activating ILC3 and dendritic cells with an enhanced Th1 response, and producing serum IgG and serum and salivary IgA specific to the administered bacterial antigens. The activated immune system also protects against other bacteria and viruses due to a trained immunity effect. Most studies show that the number of RTIs and their severity decrease in Bacterial Lysates-pretreated patients, without relevant side effects. The Bacterial Lysates treatment, in addition to reducing the number of RTIs, also prevents the deterioration of the underlying disease (i.e., COPD) induced by repeated infections. Despite these positive data, the most recent meta-analyses evidence the weakness of the studies performed, which are of low quality and have an inadequate number of patients, some of which were non-randomized while others were without a control group or were performed contemporarily in different clinical conditions or with different ages. The high heterogeneity of the studies does not allow us to state Bacterial Lysates’ effectiveness in preventing RTIs with sufficient certainty. To completely define their indications, double-blind, placebo-controlled, multicenter, randomized clinical trials should be performed for each product and for each indication. The study population should be adequate for each indication. For this purpose, an adequate run-in phase will be necessary.

1. Introduction

Respiratory tract infection (RTI) is defined as any infectious disease of the upper or lower respiratory tract. Upper respiratory tract infections (URTIs) include the common cold, laryngitis, pharyngitis/tonsillitis, acute rhinitis, acute rhinosinusitis, and acute otitis media. Lower respiratory tract infections (LRTIs) include acute bronchitis, bronchiolitis, pneumonia, and tracheitis. RTIs are the most prevalent infectious diseases and cause millions of deaths annually worldwide [1]. Further, respiratory infections are the main comorbidity factor in chronic obstructive pulmonary disease (COPD), chronic rhinosinusitis, and asthma. In total, 17 billion URTI incident cases [2] and 489 LRTIs [3] were estimated in 2019 globally, with a relevant social impact. In fact, RTIs provoke a high disability-adjusted life-years rate, with a descending trend with increasing age in children and an increasing trend with age in adults [2,3]. The Global Burden of Disease Study in 2016 reported 2,377,697 deaths in people of all ages worldwide, of which 652,572 occurred in children (<5 years of age) and 1,080,958 occurred in adults older than 70 years [4].

The highest incidence of infection observed in early life, in particular in preterm infants, is due to immature immune responses during the transitional postnatal period [5,6,7,8]. The age-related immune dysfunctions associated with inflammaging and the presence of comorbidities contribute to the increased risk of morbidity and mortality from RTIs among the elderly [9,10,11,12]. Moreover, the elderly have a weak response to vaccines and their innate immune system is less able to clear infections [13,14]. The presence of RTI has been reported to increase mortality in old people by 6–7% [15]. Independently of age, chronic airway diseases such as asthma, COPD, or cystic fibrosis are associated with recurrent RTIs (rRTIs), with worsening of their conditions [16,17,18,19,20]. Active and passive smoking [21,22], low serum vitamin D levels [22,23], a lack of physical activity [24], sudden changes in temperature at work [25], occupational exposure to various physical, chemical, and other hazards [26,27], obesity, and Type 2 diabetes [22,28,29] are reported as risk factors for RTIs. Moreover, immunocompromised individuals (including those with immunodeficiency, cystic fibrosis, and HIV, as well as the use of corticosteroids, transplantation, and post-splenectomy) [30,31,32,33,34,35,36,37,38,39,40] and those with anatomical anomalies including facial dysmorphic changes [41] or nasal polyposis [42] are also at high risk for RTI.

The use of antibiotics remains the most efficacious treatment of bacterial RTIs and accounts for 60% of all antibiotics prescribed [43,44,45]. Antibiotic use, in addition to hygiene measures, could reduce under-5 deaths in countries with the highest LRTI burden [3]. However, despite antibiotics, bacterial infections are still a severe health problem because of the emergence of resistant bacteria, antibiotic overuse and misuse, and the lack of new drug development [46,47]. Antibiotic resistance leads to higher medical costs, prolonged hospital stays, and increased mortality. On the other hand, there is evidence that antibiotics have limited efficacy in treating a large proportion of RTIs [48], including acute otitis media, acute cough/acute bronchitis, acute sore throat/acute pharyngitis/acute tonsillitis, acute rhinosinusitis, and the common cold. Moreover, in an international prospective study, van Houten et al. [49] evidenced major antibiotic overuse in patients with RTIs due to viral infections in adults. The authors underlined the need for effective interventions to decrease antibiotic overuse in RTI patients of all ages. The inappropriate prescribing of antibiotics has the potential to cause drug-related adverse events (diarrhea, nausea and vomiting, dizziness, yeast infections, hypersensitivity, and adverse reactions to antibiotics).

Prevention should be the key intervention to address RTI incidence and the frequent consequent disabilities and mortality, with vaccines being the most promising strategy [3,50]. However, there is still a lack of vaccines against most of the infectious agents responsible for RTIs, so other prophylactic strategies must be developed [51,52]. Bacterial Lysates were introduced in the 1970s as oral vaccines for the prevention and treatment of RTIs [52,53,54] and are currently used in many countries worldwide.

In September 2019, the European Medicines Agency stated that there is “… some evidence of effectiveness of these medicines in the prevention of recurrent respiratory tract infections and the safety profile is in line with what is expected for this type of product”. The Committee for Medicinal Products for Human Use therefore recommended that use of BLs for prevention can continue, but the companies must provide further data on safety and effectiveness from new clinical studies by 2026” (clinical studies were postponed by 2 years due to the COVID pandemic) [55].

In this review, the authors summarize the existing literature concerning the mechanisms of actions and the clinical efficacy of Bacterial Lysates, reporting both the substantial homogeneity of the positive results and the weakness of the studies performed.

2. Bacterial Lysates—Definition, Main Bacterial Species, and Processing Methods

Bacterial Lysates are mixtures of antigens derived from inactivated pathogens frequently involved in RTIs. The European Medicines Agency defines Bacterial Lysates as medicines made from bacterial cells that are broken down and are intended to stimulate the immune system to recognize and fight infections.

Bacterial lysis is obtained by chemical (alkaline) or mechanical treatment, while heat and detergents have also been used. The antigenic structure seems to be better maintained by mechanical disruption, while chemical lysis produces denaturation of bacterial antigens. Obtained antigens are then mixed in specific proportions to obtain a polyvalent Bacterial Lysate [56]. Commercially available Bacterial Lysates are listed in

Table 1. Characteristic commercial Bacterial Lysates used for treating respiratory tract infections.

Alkaline lysis
Broncho-Vaxom (OM-85 BV)	Haemophilus influenzae Klebsiella pneumoniae Klebsiella ozaenae Moraxella catarrhalis Staphylococcus aureus Streptococcus pneumoniae Streptococcus pyogenes Streptococcus viridans	Oral capsules
Liuvac LW50020	Haemophilus influenzae Klebsiella pneumoniae Moraxella catarrhalis Staphylococcus aureus Streptococcus pneumoniae Streptococcus pyogenes Streptococcus mitis	Oral Tablets
Lantigen B	Haemophilus influenzae Klebsiella pneumoniae Moraxella catarrhalis Staphylococcus aureus Streptococcus pneumoniae Streptococcus pyogenes	Sublingual Drops
Mechanical lysis
Ismigen PBML	Haemophilus influenzae Klebsiella pneumoniae Klebsiella ozaenae Moraxella catarrhalis Staphylococcus aureus Streptococcus pneumoniae Streptococcus pyogenes Streptococcus viridans	Sublingual tablets
Ribomunyl D53 Ribosomal–proteoglycan	Haemophilus influenzae Klebsiella pneumoniae Streptococcus pneumoniae Streptococcus pyogenes	Oral tablets or granules

. The various procedures for lysis, not completely available to the research community, should be appropriately reported and studied to allow the scientific community to verify the possible antigenic alteration. On the other hand, there are no standardized protocols for the various methods of lysis, and this makes their reproducibility challenging, with difficulty in evaluating the class effect of the various preparations and comparing the immunological and clinical effects of Bacterial Lysates.

3. Mechanisms of Action of Bacterial Lysates

The rationale for using Bacterial Lysates for the prevention of rRTIs lies in the hypothesis that they mimic natural exposure to microbes, promoting an innate, adaptive, antigen-specific immune response against the administered antigens. Bacterial Lysates can be administered orally, intranasally, or sublingually; the latter two directly activate local mucosal immunity at the level of the respiratory epithelium and submucosa [57]. The activity of the first route lies in the known interaction between the intestinal and bronchial immune system (the so-called gut–lung axis) [52,58,59,60,61]. Immune cells activated in the intestinal mucosa migrate through mesenteric lymph nodes, blood, and lymph to the lymphoid tissue of the bronchial mucosa. Bacterial antigens administered through Bacterial Lysates act as pathogen-associated molecular patterns on innate immunity and as antigens on adaptive immunity [60,62]. Bacterial Lysates prime the immune system, activating antigen-presenting cells such as macrophages and dendritic cells (DCs) and epithelial cells through TLR-2 and TLR-4 receptors [62,63,64,65,66,67]. As a consequence, mature-activated DCs produce IL-1, IL-6, and TNF-α (inflammatory cytokines) [63] and secrete IFN-α, IFN-γ, IFN-β [68], IL-8, CCL2, and CCL20 [69] (inflammatory chemokines). This results in increased cytotoxicity, in particular with an increase in CD8 cells, and the activation of NK cells and phagocytic activity [66,67]. Moreover, BL-activated alveolar macrophages produce Il6, Il1b, TNF-α, inducible nitric oxide synthase, and antimicrobial peptides Lcn2, CAMP, and Saa3 [70]. DCs are also essential players in adaptive immunity, acting as antigen-presenting cells. BLs/TLR binding activates DCs, resulting in the up-regulated expression of MHC II, CD40, CD86, and HLA-DR and the de novo expression of CD83. DCs migrate to lymph nodes where they interact with T cells [71,72,73,74,75]. This is followed by the activation of T cells and differentiation of B cells into plasma cells, with the increased production of antigen-specific immunoglobulins [74,76,77]. In particular, it has been shown that intranasal Bacterial Lysate administration enhances IgA concentrations in the respiratory mucosa, with consequent protection against respiratory microbes [78,79,80]. The BL-induced immune response has a Th17/Th1 profile, with increased production of IL-17A and IFN-γ and decreased IL-4 levels [63,70].

The activation of innate immune cells allows protection against other bacteria and viruses in addition to those specifically administered with Bacterial Lysates. BL-activated bronchial and alveolar epithelial cells participate in this immune protection [81]. In fact, BL-stimulated alveolar and bronchial epithelial cells highly express several pro-inflammatory chemokines (Cxcl1, Cxcl8, Ccl20, Cxcl11, Ccl2, and Ccl5) and cytokines (IL6 and TNF-α) [70] and the novo expression of beta defensin 1, a major antimicrobial peptide [70,82]. Moreover, BL-stimulated bronchial epithelial cells release IL-23, which induces IL-22 production by innate lymphoid cells, in turn favoring the release of antimicrobial peptides by bronchial epithelial cells [82].

Bacterial Lysate administration also has anti-viral activities, inducing both DCs to produce anti-viral cytokines/chemokines (IFN-β, IFN-γ, IL-8, CCL2, CCL20, and TNF-α) [83] and polyclonal activated B cells to produce IgA and IgG specific to the influenza virus and the respiratory syncytial virus in animals not previously exposed to these viruses [74]. Increases in virus-specific IgA and IgG were also seen in children affected by bronchiolitis after treatment with Bacterial Lysates [84]. Moreover, BL-activated NK cells are involved in the elimination of virus-infected cells [85]. On the other hand, it has been demonstrated that treatment with Bacterial Lysates preventing RTI inhibits the “vicious circle” of bacteria/virus interaction [86].

Some authors have suggested that taken together, the activation of the innate and adaptive immune system generates a state of “pre-alert” against infection [80]. It is the concept of “trained immunity-based vaccines” that generates a strong immune stimulus, which increases the nonspecific response of innate immune cells and strengthens the activated state of dendritic cells to enhance adaptive T cell responses to specific and bystander antigens [87,88]. Figure 1 summarizes the immune mechanisms of Bacterial Lysates.

The lungs have tertiary lymphoid structures, bronchus-associated lymphoid tissue (BALT), which consists of lymphoid follicles in the lungs and bronchus. BALT is an effective priming site of adaptive B cell and T cell responses directed against airborne antigens. It consists of a stromal cell network, with separated areas for T and B cells, endothelial venules, and lymphatics [89]. Physiologically, BALT is present in the lungs of children to give protection to the diverse respiratory challenges during this formative life stage. In adults, BALT has not been reported in healthy adults but is inducible (iBALT) under pathologic conditions, such as chronic or repeated respiratory infection [90]. IBALT forms in the perivascular space of blood vessels on the basal side of the bronchial epithelium, and consists of a B cell follicle with T cells and DCs surrounding the B cell follicle [91]. There are no specific studies on the direct activity of Bacterial Lysates on BALT or iBALT, as they can be administered only by the oral route (sublingual or gastrointestinal). However, even though the various tertiary lymphoid structures (gastrointestinal, nasal, laryngeal, and pharyngeal) are separated, they communicate through the common mucosal immune system [92]. It follows that lymphocytes induced by a specific antigen at one site can migrate as effector cells, providing protection from the same antigen in other organs. This is understood as the concept of an “integrated immune system” [93].

4. In Vitro and Animal Experiments

Research in animals has revealed that Bacterial Lysates act as immunomodulators. Blesser et al. [94] found that Bacterial Lysates in mice increase the production of immunoglobulin specific to the bacterial strains contained in the extract. In the same experiments, all BL-pretreated mice survived after being infected either by Haemophilus influenzae virus or Salmonella typhimurium, with respect to non-pretreated mice. Moreover, in a further experiment, they found that 70% of mice pretreated by Bacterial Lysates and then exposed to a sublethal dose of influenza virus and superinfected by S. pneumoniae survived, whereas all non-pretreated mice died after 3 days [95]. Bacterial Lysates have also been evidenced to protect against pneumococcal pneumonia, engaging pro-inflammatory gene expression in macrophages and epithelial cells [70]. Both Bacterial Lysates obtained by chemical or mechanical lysis act as TLR antagonists, inducing NF-kB activation in a MyD88-dependent manner and inducing antibacterial activity in mice lungs [64].

Pretreatment with Bacterial Lysates in primary human bronchial epithelial cells of healthy, asthmatic, and COPD patients infected by rhinovirus reduced the number of infected cells with an increase in their survival with respect to non-treated cultures. Bacterial Lysate activity was supported by the concomitant activation of Erk1/2 MAPK and cAMP signaling in epithelial cells along with an increase in C1q-R and β-defensin [96]. In addition, in murine models, Bacterial Lysates positively modulate the production and release of inflammatory cytokines and the antibody response against both respiratory syncytial virus [97] and influenza virus [74,97] through the activation of DCs via TLR4 [97]. It has been demonstrated that in human immune cells, TLR2 and TLR4 orchestrate the immune response to a Bacterial Lysate, which binds to both receptors [98]. In a further study, a preventive BL airway administration inhibited viral replication in a dose- and time-dependent manner, with an increase in IFN-β gene expression, also preventing the inflammation and limiting the ILC2 recruitment in the airways [99]. Recently, it was evidenced that Bacterial Lysates significantly downregulated ACE2 transcription and expression, inhibiting the SARS-CoV-2 infection of bronchial epithelial cells [100]. Furthermore, there is evidence in animal experiments that Bacterial Lysates induce the prevalence of the Th1/Th17 response with respect to the Th2 type [70] and induce an increase in the expression of T regulatory cells [80,101]. These changes in T cell prevalence suggested the possibility of counteracting the Th2 inflammation typical of an allergic reaction with the administration of Bacterial Lysates. In fact, in animal experiments, Bacterial Lysates prevented allergic sensitization through the activation of IL10-producing T regulatory cells [101,102] and reducing the level of IgE [103]. Moreover, interference with the airway epithelium/IL-33/ILC2 axis and lung allergen-induced TH2 response, along with the induction of tolerogenic dendritic cells, was the underlying mechanism of suppression of alternaria-induced experimental asthma by preventive/concurrent Bacterial Lysate administration during sensitization in mice [104]. Moreover, other authors also evidenced a significant reduction in eosinophilic infiltration, mucus plugs, and airway wall thickness in a model of asthmatic mice after oral administration of Bacterial Lysates with respect to non-treated mice [105].

5. Clinical Efficacy of Bacterial Lysates

Clinical trials with Bacterial Lysates, obtained via both chemical and mechanical lysis, were carried out in patients particularly susceptible to RTIs, such as children, the elderly, and COPD patients. A reduction in infection episodes was found in most studies as a clinical consequence of the Bacterial Lysate immunostimulatory effects.

5.1. Pediatric Patients

The first trials were conducted in the 1980s and 1990s. All but one showed a reduction in infectious episodes in BL-treated children. In a double-blind clinical trial, the incidence and duration of infectious episodes (and the duration of concomitant treatments) significantly decreased in Bacterial Lysate-pretreated children with rhinosinusitis with respect to controls. The authors evidenced a correlated increase in serum IgA [106]. The risk of suffering from three or more infectious episodes of the upper respiratory tract and one or more episodes of gastroenteritis was reduced by 40% in BL-pretreated patients, as reported in a large trial involving 423 children at high risk of infection, as they attended day-care centers [107]. A further trial evaluated the efficacy of the combination of Bacterial Lysates added to amoxicillin/clavulanate in the treatment of subacute sinusitis with respect to an antibiotic plus placebo. The infectious episodes in the active group lasted less than in the control group treated only by antibiotics, with a subsequent lower incidence of RTIs [108]. The only negative study was performed in 1986 with no significant differences in the number of acute RTIs between children (n = 825) receiving intranasal BL treatment and children in the placebo group (n = 327) [109].

In the following decades, more trials were performed, all showing the efficacy of Bacterial Lysates in preventing recurrent infections in children. A one-year RCT involving 188 children evidenced a 50% reduction in infections in treated patients with a sustained effect during the 6-month follow-up after the end of the study, without relevant side effects [110]. A further one-year RCT, on children aged 1–12 years with a history of RTIs, evidenced a significant reduction in RTIs and antibiotic use in BL-treated children with respect to the controls [111]. This trial evidenced, as a secondary endpoint, a 68% reduction in acute otitis media in BL-treated children, in agreement with a previous evaluation [112]. Similar results on the prevention of otitis media were obtained by Mora et al. [113]. The preventive effects of Bacterial Lysates in the treatment of children with asthma and RRIs were also observed by Su et al. [114] and Chen et al. [115] in China with randomized placebo-controlled trials.

The data on the effect and safety of Bacterial Lysates in reducing upper RTIs were confirmed by subsequent large RCTs [116,117,118], also showing beneficial effects in children with IgG deficiency [117]. An increase in B-lymphocytes in BL-treated subjects, concomitant with clinical benefit, was seen [118]. A reduction or no episodes of acute tonsillitis were evidenced in a 5-year retrospective cohort study on 177 children with 131 receiving Bacterial Lysates. It is of note that no patients with total success required tonsillectomy in the long-term follow-up [119].

A recent DBPC trial evidenced a significant reduction in day-care absence for children and therefore lost working days for parents, in addition to a significant benefit in the number of RTIs among BL-treated children with respect to those receiving a placebo; the effects were greater in children with a history of frequent RTIs [120]. In a 6-month prospective cohort study involving 57 children (aged 1–9 years), a decrease in the number of RTIs was evidenced, along with a decrease in the severity of symptoms in the absence of adverse effects [121].

A further aspect was evidenced in children, linked to the ability of Bacterial Lysates to influence the Th1/Th2 balance of the immune response in favor of Th1 [70] with a significantly increased release of IFN-γ and IL-12 [63,122,123], enhancing the reaction against microorganisms and weakening the Th2 type response. Moreover, Bacterial Lysates concomitantly induced the differentiation of T regulatory cells [124] that are protective against allergy and asthma. This led some authors to study the effects of Bacterial Lysates on allergy and asthma, with microorganisms, in particular a virus infection, being a trigger for asthma exacerbation and high Th2 and a defective Treg response being characteristic of the allergic sensitization [66,95,125,126]. Moreover, the respiratory syncytial virus and human rhinovirus induce airway inflammation, epithelial damage, and a type 2 immune response. Recurrent infections in children, also facilitated by their physiological immune immaturity, can induce a persistent Th2 prevalent immune reaction, therefore facilitating allergic sensitization [127,128]. The hypothesis was that the ability of BL prophylaxis to prevent recurrent wheezing/asthma attacks lies in the modulation of the immune response against viral infections [59]. The benefit of BL prophylaxis was observed in a randomized, double-blind, placebo-controlled, parallel-group study including 75 children (aged 1–6 years old) with recurrent wheezing and viral infections. The administration of Bacterial Lysates provoked a significant reduction in acute RTIs and a 37.9% reduction in wheezing episodes with respect to the placebo group (p < 0.001) [129]. These results were similar to those observed in a previous 12-month study [130] and in a study of school-aged asthmatic children on long-term control with inhaled corticosteroids [131]. These effects were associated with an increase in serum IFN-γ and IL10, demonstrating an immune Th1 deviation and an increase in T regulatory activity [132].

The efficacy of Bacterial Lysates was also evaluated in the primary prevention of lower RT infections in a recent study in at-risk children [133]. The infectious episodes were significantly less frequent and less severe in children treated with Bacterial Lysates with respect to the controls.

Based on the BL interference with the Th1/Th2 balance and their effects on T reg, a large multicenter study was started with the primary objective of evaluating the possible prevention of wheezing and asthma with Bacterial Lysate administration since the first year of life. The study is not yet completed (https://classic.clinicaltrials.gov/ct2/show/NCT02148796, accessed on 3 March 2024).

In fact, recent reviews and meta-analyses [134,135,136,137,138] have reported substantial evidence for the benefits offered by Bacterial Lysates in reducing the risk of RTIs in children, although they all revealed important weaknesses in the conducted studies as reported in the meta-analysis chapter.

5.2. Adult Patients

Bacterial and virus infections are increasingly recognized as an independent stimulus to airway inflammation [18], modulating the severity and frequency of COPD and chronic bronchitis exacerbations [139,140,141], which are the main conditions associated with recurrent RTIs. Therefore, the treatment of such infections may also be useful in delaying the evolution of these diseases. One of the first RCTs performed in 1990 [142] involving 265 COPD patients treated by Bacterial Lysates demonstrated a statistically significant reduction in infectious events and a concomitant reduction in the use of antibiotics. Also in the 1990s, double-blind, placebo-controlled trials showed the effectiveness of Bacterial Lysates in reducing the mean score of symptoms, the number, severity, and duration of acute exacerbations, and the risk and number of days of hospitalization for respiratory problems with a reduction in the use of conventional therapy in BL-treated groups with respect to placebo groups [143]. Opposite effects were seen in a very large study conducted in 1997, in which the authors reported no differences in COPD exacerbations, hospitalizations, and the severity of the disease in BL-treated patients with respect to controls [144]. During the 2000s, an RCT confirmed the activity of Bacterial Lysates on BPCO exacerbations and found a significant reduction in antibiotics use in the actively treated group with respect to the placebo group [145]. Solèr et al. [146] confirmed such results in the elderly with chronic bronchitis and COPD, further highlighting that current or past smoking patients saw greater benefits from the treatment. Cazzola et al. [147] studied the effects of the combination of Bacterial Lysates with salmeterol/fluticasone versus an inhalant alone. The trial showed a reduced total number of exacerbations, rate of exacerbations per patient per year, number of exacerbations needing oral corticosteroids, and total number of hospitalizations in BL-treated patients. Genda et al. [148] confirmed the data on exacerbations and hospitalization. In contrast, the DBPC trial [149] conducted with 288 moderate to very severe COPD patients assigned to either Bacterial Lysates or a placebo, in addition to inhalation therapy, did not achieve their primary outcome (a reduction in the number of exacerbations by 25% in the Bacterial Lysate-treated group with respect to the placebo group). However, a reduced number of days with fever, days of hospitalization, and number of days in poor health were observed in the BL-treated group. A meta-analysis published in 2015 concluded that the current evidence did not support a beneficial effect of Bacterial Lysates for COPD patients in terms of the duration of hospitalization and the severity of acute exacerbation [150]. The authors were only able to include five trials in the meta-analysis, with the majority of studies performed until then of very low quality. However, in the following years, a number of trials reported positive results on the efficacy of Bacterial Lysates in COPD patients. A clinical trial on 384 patients showed that 12 weeks of BL treatment significantly reduced the proportion of patients with acute exacerbation with respect to the controls. These benefits were maintained for up to 22 weeks [151]. Positive results were achieved by a further trial on 150 COPD elderly patients regarding the number of acute exacerbations in BL-treated patients with respect to controls. Further, there was evidence that the addition of Bacterial Lysates to the recommended treatment delayed the deterioration in lung function in elderly patients with stable COPD, with a significant difference in lung function parameters between the two groups [152]. These results on clinical and functional parameters were confirmed by an RCT on 60 patients with frequent exacerbations of COPD [153]. Ricci et al. [154] evidenced a reduction in infectious episodes after treatment with Bacterial Lysates through the induction of an antibody-mediated immune response, efficient not only against bacteria present in the product but also against different microbes. A metanalysis published in 2022, with the evidence of these most recent trials, underlined the efficacy of Bacterial Lysates in reducing the exacerbation rate, the mean number of exacerbations, and the severity of the disease in COPD patients, without relevant side effects [155]. However, the heterogeneity in addition to the other important weaknesses of the selected studies meant the data on efficacy were not conclusive (see the meta-analysis chapter).

Susceptibility to RTIs is typical not only of patients with COPD and chronic bronchitis but also of other adult categories, such as the elderly, people living in communities, and patients with immunodeficiency and other chronic pathologies.

In the 1980s and 1990s, a number of clinical studies were performed in non-BPCO adults with RTIs, demonstrating the efficacy of Bacterial Lysates in reducing the number of acute infectious episodes [146,147,148,149,150,151,152,153,154,155,156,157,158] and the number, severity, and duration of upper and lower RTIs [159]. Subjects with purulent sinusitis had lower numbers of infections when pretreated by Bacterial Lysates, with reduced purulent nasal discharge, coughing, and headaches [160]. Positive results on RTIs were also achieved in subjects receiving hemodialysis with a significant protective effect [161]. In the 2000s, an RCT involving 140 adults with RTIs evidenced better effects of Bacterial Lysates compared to placebo on the number and duration of RTIs, working days lost because of infections, and the need for antibiotics. In this study, Bacterial Lysates obtained via mechanical lysis were more efficacious in comparison to those obtained by chemical lysis [162]. A successive study also showed a reduced duration of infectious episodes and a decreased need for antibiotics in patients with autoimmune nephrosis [163]. In an interventional, non-randomized study, treatment with Bacterial Lysates was followed by a reduction in RTI with respect to prior treatment, with reduced need for the use of antibiotics in 104 HIV-infected patients [164]. An interesting study was performed in cloistered nuns with recurrent URTIs. A significant reduction in the number and duration of infections was observed in the active treatment group with respect to the placebo, with an increase in salivary IgA in the active treatment group only [165]. Braido et al. [166] performed a double-blind, placebo-controlled, randomized multicenter trial involving 160 patients (71 in the active group and 81 in the placebo group) with a mean age of 42.4 ± 15.14. Both the primary (the number of RTIs) and secondary endpoints (RTI duration, frequency and severity of the acute episodes, the use of drugs, and the number of missed workdays) were reached, with a high statistical significance compared with the placebo group. The estimated risk of needing antibiotics was reduced by 52.1%. The only limitation of the study was that no discrimination was made between URTIs and LRTIs.

A recent study explored the effect of Bacterial Lysates in the treatment of patients with non-severe community-acquired pneumonia. A group of patients received antibiotic treatment alone, and in the second, third, and fourth groups with Bacterial Lysates, an immunomodulator (azoximer bromide) or placebo was added. The overall duration of all symptoms was lower in the BL/immunomodulator groups compared with the control group, with an improvement in the pro-inflammatory cytokine profile [167]. The clinical benefit lasted the successive 2 years of follow-up.

The work of Pizzimenti et al. [168] is of great interest, which found that a Bacterial Lysate, Lantigen B, modulated the expression of ACE2, hypothesizing the possibility of administering Bacterial Lysates for the prevention of SARS-COV.

6. Meta-Analyses on Bacterial Lysates

Here, we report the three most recent meta-analyses published in 2022/2023, the first on the effect of Bacterial Lysates in children [138], the second in adults with COPD [150], and the last specific to one Bacterial Lysate, Lantigen B [169].

The meta-analysis on the effects of Bacterial Lysates in children [138] found only 38 trials with sufficient quality to be evaluated: all but 3 (2017, 2019, and 2020) were performed before 2004, the majority in the 1980s and 1990s. The quantitative analysis indicates that Bacterial Lysates reduce RTI incidence in children by 40% on average, with this observation constant across all studies. However, the authors reported low-quality evidence, substantial heterogeneity, and the possibility of publication bias. The quality of the evidence (GRADE) was low for the number of RTIs and the ratio of mean RTIs, with high heterogeneity in the number of RTIs in the control group, and very low on the adverse effects, which were reported in only 14 trials. Moreover, various studies were performed with subjects with a wide age range (aged 1–18 years) and both URTIs and LRTIs were included. Most studies lasted <6 months. Moreover, the primary endpoints of the trials were diverse. Only 19 trials were conducted with more than 40 patients.

The meta-analysis on the effect of Bacterial Lysates on COPD [155], reporting Bacterial Lysates as useful in reducing exacerbations and symptoms, was based on only twelve studies (nine randomized controlled trials and three abstracts of randomized controlled trials with sufficient data) performed from 1998 to 2020 with sufficient scientific quality, showing a significant clinical efficacy of Bacterial Lysates overall. The duration of the treatment lasted from 12 weeks to 6 months in the various studies, and the follow-up period was 10 weeks to 9 months. Six studies did not report dropouts. The two main biases were the differences in lung function, correlated with exacerbation risk, and the non-standardized symptom evaluation. The severity of COPD varied greatly among the studies with the mean FEV1/predicted ranging from 30% to 85%. Only eight studies reported the rate of exacerbation with detailed numbers and only four reported the hospitalization rate. Moreover, the selected studies did not homogeneously report the efficacy against symptoms (the changes in sputum, cough, severity of dyspnea, and fever). Furthermore, the included studies also had methodological issues such as explicit allocation concealment, which can reduce the strength of the evidence. Finally, some important issues were not evaluated: are Bacterial Lysates effective in preventing exacerbations of COPD in high-risk patients with severely impaired lung function and reducing the need for antibiotics and corticosteroids?

All possible literature on the effects of a single Bacterial Lysate (Lantigen B) was analyzed by Braido et al. in 2023 [169] who obtained the manuscript of the oldest studies, studies published in non-indexed national journals, and unpublished data from the manufacturer. In total, 37 studies were analyzed, of which 22 were eligible for the meta-analysis. A total of 4571 patients (of which 2888 were from a single study conducted in 1981) were evaluated in these studies, with 2421 treated with Lantigen B and 2150 controls. Fourteen studies were performed in adults, seven in children, and one in both. Eight studies were performed in healthy subjects, with the others involving patients with recurrent RTIs. The quantitative analysis showed that all primary outcomes (the number of exacerbations during the study period, days of illness for recurrent RTIs, number of days with fever, and number of days absent from work or school) were met in patients treated with the Bacterial Lysate with respect to the placebo group, with an overall reduction of −47%, evaluating all studies together. The data were similar considering healthy subjects, adults with or without recurrent RTI, and pediatric patients separately. Moreover, the prophylactic effect was lower in healthy subjects with respect to patients with a history of recurrent RTIs.

However, these results should be interpreted with caution. In the qualitative analysis, the authors pointed out the significant heterogeneity of the study methods and the accuracy of the reports. The small sample size of the majority of the studies makes it difficult to analyze the effect of the Bacterial Lysate in different pathological conditions predisposing patients to RTIs. Not all studies shared the same primary outcomes, the follow-up varied from 1 to 7.5 months, and not all studies reported/described the determination of the sample size, randomization procedure, number of dropouts, accurate statistical analysis, and adverse events. These limitations and the high heterogeneity of the studies make it impossible to draw definitive conclusions on the clinical efficacy of Bacterial Lysates in the prevention of RTIs in both adults and children. However, there is strong homogeneity in the observed results despite the large standard errors observed. The authors concluded that, with the frequencies of responses being very constant across the various studies, the clinical efficacy of Lantigen B in RTI prophylaxis in both adults and children with recurrent RTI is highly suggestive.

7. Discussion

This analysis of the literature related to the efficacy of Bacterial Lysates allows us to conclude that the data published are in favor of the use of Bacterial Lysates for the prevention of RTIs. The various published reviews focused, in general, on Bacterial Lysates as a class, others on specific Bacterial Lysates, others on RTI prevention in general, and finally, a portion on specific clinical conditions favoring RTIs such as BPCO, immune system immaturity in children, and immune system failure in elderly.

In addition to the partial positive note on clinical efficacy, all authors agreed that there are severe limitations among the trials performed, so there is a need for high-quality, large, multicenter, double-blind, placebo-controlled randomized clinical trials to confirm the role of immunostimulants in preventing RTIs in children as well as in adults, elderly, and COPD patients.

One of the main problems to be addressed in future studies is the determination of the sample size because, after the COVID-19 pandemic period, the number of RTIs has been reduced worldwide with respect to previous periods [170,171,172]. Therefore, there is a need for a correct evaluation of the real incidence of RTIs: the lower the RTI incidence, the higher the sample size of the study.

The trials should be made in selected specific clinical conditions and age groups to allow a well-defined therapeutic indication of the tested product.

Bacterial Lysates can be considered a class of drugs but there are many differences among the various products. Bacterial Lysates have different bacterial strains and are prepared by different methods with consequent diversity in the quality and purity of the product with the presence or absence of other cell constituents such as polysaccharides. Therefore, although different Bacterial Lysates may share some common mechanisms, some results may be specific to certain products.

As we found no study comparing all Bacterial Lysates, it was not possible to establish the superiority of a certain kind of pharmaceutical preparation. It will be useful to compare products with the same bacterial culture made using different procedures. This would allow for choosing the best method for preparing immunostimulant lysates. Moreover, there were no studies comparing the efficacy of the different routes of administration: local (nasal and sublingual) and oral.

Finally, there are insufficient data on the use of Bacterial Lysates in acute infections, immunodeficiencies, autoimmune diseases, and active tuberculosis and cardiopulmonary insufficiency.

In any case, specific vaccines remain the first choice for some bacteria, and an example is that against Streptococcus pneumoniae. The are four vaccines for this bacterium, differentiated by the number of serotypes they contain. It has been reported that 60% to 70% of the success in preventing pneumococcal disease is caused by serotypes present in the vaccine [173]. Even this vaccine, as with others, induces a trained immunity against other infections. The effectiveness of a vaccine is therefore higher than that observed with Bacterial Lysates. However, there are some issues to be considered. Vaccination is often followed by an increase in the frequency of infections by serotypes not present in the administered vaccine. Furthermore, it has been reported that there is an increase in antibiotic resistance among nonvaccine serotypes [174].

Furthermore, it will be interesting to study the effects of Bacterial Lysates with molecules stimulating the immune system, such as FLT3L and MALP-2. The first acts as a growth factor on B and T cells by activating the hematopoietic progenitors [175]. It has been shown that Flt3L induced an increase in the number of dendritic cells in nasal-associated lymphoid tissue [176]. The second stimulates the response of macrophages, indirectly activates NK cells, and enhances antigen presentation on dendritic cells [177].

8. Conclusions

The present review underlines the major shortcomings of the studies performed on the effects of Bacterial Lysates in the prevention of RTIs. It appears evident that the lack of rigor in experimental design and standardized protocols, insufficient patient numbers, or other technical faults have led to some level of mistrust in the clinical trials on the use of Bacterial Lysates in a clinical setting.

High-quality, large, multicenter, double-blind, placebo-controlled, randomized clinical trials should be performed to establish the actual effects of immunostimulants and the effects of individual immunostimulant preparations, selecting patients by age, risk of RTI, comorbidities, and route of administration.