Adjuvant activity of glucosaminylmuramyldipeptide and prospects of its use for the treatment and prevention of infectious diseases

Introduction

To date, a fairly large number of immunomodulatory drugs are used in clinical practice for the treatment and prevention of many infectious and non-infectious diseases. The most promising are adjuvants, which, when co-administered with attenuated or killed antigens, can increase the immune response of the macroorganism, but at the same time be weakly reactogenic [1]. In the course of numerous studies, it was found that substances of natural origin obtained using chemical synthesis methods were the most effective and safe immunomodulators and adjuvants. A promising class of synthetic immunomodulators is muramyldipeptide (MDP) and its derivatives, including glucosominylmuramyldipeptide (GMDP). The advantage of these drugs is a sufficiently well-studied chemical composition and structure of the molecule of substance, an easily reproducible structure, and the absence of harmful and undesirable impurities [2].

Approximately 100 literature sources related to the use of muramyl dipeptides from the RSCI, eLibrary, and MedLine databases over the past 20 years were analyzed for this review. The review cites 52 of the most significant papers covering the study of the use of GMDP mainly for prophylactic purposes in infectious diseases, as well as the adjuvant effect of the drug. The review did not include work on the use of GMDP in other various fields of medicine, as well as the use of foreign analogs of GMDP as an adjuvant, such as murabutide, romurtide, and 6-O- (tetradecylhexadecanoyl) -N-acetylmuramyl-L-alanyl-D-isoglutamine.

Discovery history of the drug

MDP was isolated from Mycobacterium tuberculosis by French scientists in 1974. This compound, consisting of one sugar molecule and two amino acids, is a component of peptidoglycan of the bacterial cell wall and has immunogenic and adjuvant activity [3].

At the same time, MDP was obtained from the Lactobacillus bulgaricus bacterium under the guidance of Professor V.T. Ivanova and Dr. T.M. Andronova, and contained the second sugar molecule N-acetylglucosamine, unlike a foreign substance. This compound, called GMDP, demonstrated not only higher adjuvant activity but also the ability to stimulate non-specific resistance to bacterial and viral infections, while being a non-toxic and slightly pyrogenic substance. Soviet scientists developed a chemical method for producing highly purified GMDP with standard characteristics on an industrial scale and, most importantly, with predictable bioactivity. Subsequently, this drug successfully passed clinical trials in leading medical institutions in Russia, Australia, and the UK and received state registration [4].

Employees of the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of RAS and the Institute of Immunology of the FMBA of Russia in 1996 were awarded the State Prize in the field of science and technology for the development and creation of a biotechnological production of a new immunocorrective drug. Currently, GMDP is widely used in Russia, in the republics of Belarus, Georgia, Kazakhstan, Moldova, Armenia, Azerbaijan, Kyrgyzstan, and Uzbekistan, as well as in Mongolia [3]. Extensive experience in the use of this drug has been accumulated; its effectiveness and safety for the treatment and prevention of various infectious, autoimmune, allergic, and oncological diseases, as well as secondary immunodeficiencies have been proven [4].

GMDP is recommended for the prevention of acute respiratory viral infections and other infectious diseases, increasing the effectiveness of antibacterial and antiviral drugs, the body's general resistance to pathogens, reducing the manifestation of chronic diseases during their exacerbation, as well as an adjuvant in licensed and new experimental vaccine preparations. At the same time, GMDP is a highly purified, non-toxic, non-carcinogenic [5], and non-mutagenic drug and has a simple and convenient dosage form, which allows it to be used in all age groups, including children from birth [6][7].

Since the discovery of this drug, rare cases of side effects such as fever (to subfebrile values) and diarrhea have been recorded; in isolated cases, myalgia and arthralgia have been recorded. It should be noted that all observed adverse reactions were indicated in the instructions for GMDP use [8].

Mechanism of muramyldipeptide action

GMDP is a synthetic analog of MDP, which is the minimal component of peptidoglycan of cell walls of both gram-negative and gram-positive bacteria, and is capable of causing an immune response in animals. MDP has all the necessary properties of pathogen-associated molecular structures, which is expressed in the stimulation of innate immunity factors and the ability to form protection against various foreign agents [7][9]. Normal intestinal microflora serves as a constant source of MDP, thus contributing to the maintenance of the immune system in an active state and constant readiness to protect against foreign influences. Peptidoglycan fragments are released from the cell wall of bacteria, absorbed in the small intestine, and affect the immune and inflammatory response in the gastrointestinal tract, as well as in other organs. As a result of the interaction of MDP with resident antigen-presenting cells, activated antigen-presenting cells are formed which have a homeostatic effect and promote the proliferation of regulatory CD4 lymphocytes that do not cause inflammation. Thus, normally in the human body, there is a constant stimulation of the immune system by MDP from the intestine [10].

The biological activity of MDP is carried out due to a specific interaction with the intracellular receptor protein NOD2, which is expressed in the cytoplasm of phagocytes-monocytes, neutrophils, dendritic and epithelial cells [3][11–12]. However, in addition to NOD2, other cytosolic receptors of innate immunity were identified: YB1 [13], Pept2 and Pht1 [14], the binding of MDP to which led to cell proliferation and differentiation, their response to stress exposures.

The NOD2 receptor is a member of the family of cytosolic proteins (NLR), components of the innate immune system belonging to the so-called image-recognizing receptors. The NOD2 consists of three domains: the C-terminal region is responsible for ligand recognition (MDP), and the central domain is responsible for nucleotide-binding oligomerization and the N-terminal domain (activation and recruitment of caspase) transmits information to signaling molecules and transcription factors in the nucleus [15][16]. It has been proven that activation of the NOD2 receptor is a key moment for triggering adaptive immunity to bacterial, viral, and parasitic infections, and contributes to the formation of an adequate and prolonged immune response [17]. Bacteria are absorbed by phagocytic cells, the bacterial wall is split under the influence of enzymes, and MDP is released into the cell. It binds to the ligand-binding domain of the NOD2 protein, transmits a signal from the effector domain, and triggers a cascade of reactions. The activated NOD2 receptor oligomerizes, forms an inflammasome, and interacts with protein 2 kinase, then through phosphorylation and ubiquitination reactions the signal goes to the nuclear transcription factor NF-kB (nuclear factor κB), which induces the expression of a large number of genes, in particular pro-inflammatory cytokine genes, adhesion molecules, acute-phase proteins, inflammation enzymes (NO synthase and cyclooxygenase), major histocompatibility complex molecules, etc. All these events lead to the synthesis of a wide range of cytokines IL-1, IL-2, IL-6, IL-8, IL-12, TNF-α, INF-γ, and colony-stimulating factors (CSFs) by the cell. The end result of activation is an inflammatory reaction of the body mediated by the synthesis of proinflammatory cytokines and chemokines by cells of the innate immunity system. The synthesis of IL-1 and TNF-α under the influence of MDP enhances the antimicrobial function of neutrophils, monocytes, and macrophages, increases the cytotoxic activity of phagocytes, NK cells, and killer T cells, and induction of CSF synthesis activates leukopoiesis [7][18–21].

MDP is characterized by running at once two activation paths of NF-κB (classical and alternative), which contributes to the initiation of both innate and adaptive immunity [22]. The ability of MDP to interact with NOD2 receptors of epithelial cells, inducing the synthesis of IL-6, chemokine CXCL-8, and antimicrobial peptide β-defensin, as well as increasing the production of reactive oxygen species in epithelial cells, thereby contributing to the protection of mucous membranes from pathogenic microorganisms, is equally important [23]. It should be noted that MDP activates not only the NF-κB signaling pathway, but also the MAPK (mitogen-activated protein kinase) and interferon (IFN) pathways, which leads to an increase in the synthesis of proinflammatory cytokines, induction, proliferation, and differentiation of immunocompetent cells [7][23].

Effect of the drug on the factors of innate and adaptive immunity in various infectious diseases

It has been shown that GMDP, being a synthetic analog of MDP, in the peripheral blood of the examined patients causes an increase in the total number of dendritic cells (DCs) and a statistically significant increase in the expression of the chemokine CCR7 responsible for DC migration to secondary lymphoid organs [24]. GMDP increases the expression of HLA-DR antigens, thereby improving the recognition of pathogen antigens, as well as the production of pro-inflammatory mediators, cytokines, and their soluble and membrane-associated receptors [25]. GMDP acts on monocytic-macrophage cells, stimulating the activity of lysosomal enzymes and the formation of reactive oxygen species, and acts on the absorption and cytotoxic properties of virally infected and tumor cells [7][25–31]. The use of GMDP leads to an increase in the level of intracellular defensins in the neutrophils of peripheral blood [32], an increase in their phagocytic activity, as well as bactericidal and metabolic activity [33–34].

The drug not only stimulates the bactericidal and cytotoxic activities of neutrophils and macrophages but also enhances the functional activity of effectors of the adaptive immune response in patients infected with pathogens of various etiologies.

In patients whose treatment regimen includes GMDP, recovered parameters of immune status are observed [28]: an increase in the number of CD3⁺, CD4⁺, CD8⁺, and CD16⁺ lymphocytes [26][27][31][33][35][36-38], as well as CD19⁺ lymphocytes [29][39]. During treatment, under the influence of GMDP, the concentration of the pro-inflammatory cytokines IL-1 β, IL-6, IL-8, and TNF- α, as well as the anti-inflammatory cytokine IL-4, increases [29].

In addition to stimulating cellular immunity in patients with viral and bacterial infections, an increase in the production of serum IgG [26], as well as secretory IgA, in the oral cavity was found [27][40].

Thus, GMDP triggers the mechanisms of the innate immune response, cellular and humoral immunity, effectively activating antibacterial and antiviral defense [25], which makes it possible to use this drug widely in the treatment and prevention of various infectious diseases. Despite the data obtained during clinical trials on the ineffectiveness of the use of GMDP in the prevention of influenza in the elderly [41], the presence of immunostimulatory and antiviral activity of this drug against influenza [35] and other viral infections in children and adults has now been proven [24][26–28][38–39][42][43]. The feasibility of the inclusion of an immunomodulatory GMDP drug in the treatment of patients with bacterial infections [29–33][36][40][44], including tuberculosis in adults and children [37], as well as in the complex therapy of pneumonia and urinary system infections in newborns and premature children, has been shown [7].

Positive results were obtained during experiments to study the possibility of using this immunomodulator in treatment regimens for intestinal [34] and especially dangerous infections [45].

Prospects for using GMDP as an adjuvant

In addition to using GMDP for the treatment of various diseases, the high adjuvant activity of this drug was also noted. The adjuvant effect of GMDP is due to its ability to increase the activity of neutrophils, DCs, and macrophages, increase the expression of receptors on the surface of these cells for chemotaxis, adhesion, and absorption of antigens, stimulate the processing and presentation of antigens included in the vaccine preparation, thus enhancing antibody production, antibody-mediated cytotoxicity, and other cellular immunity reactions [6]. The efficacy of the combined effect of GMDP, a number of other immunomodulators, and specific vaccines against arbovirus infections has been proven [46-47]. The ability of GMDP to enhance humoral and cellular immune response to corpuscular and soluble antigens was revealed. The presence of a pronounced stimulating effect was shown in preclinical studies of an experimental HIV vaccine containing recombinant gp120 antigen when using GMDP as an adjuvant [6].

The feasibility of introducing GMDP into the scheme of emergency and specific prevention of especially dangerous infections is evidenced by the positive results obtained by domestic scientists in improving vaccines against anthrax infection [45], brucellosis [48], as well as cholera [49–50]. The use of GMDP as an adjuvant indicates the promise of its use to increase the effectiveness of specific prevention of these infections.

Conclusion

Thus, all of the mentioned above proves the efficacy of using GMDP in the treatment and prevention of various infectious diseases, including especially dangerous ones. The ability of GMDP to activate the mechanisms of innate and acquired cellular and humoral immunities and to stimulate both antibacterial and antiviral protection opens up new prospects for its use to improve the treatment and prevention of various diseases, as well as the use of this drug as an adjuvant with licensed and experimental vaccines.