Published Time: 2024-02-02
The underdevelopment of adjuvant discovery and diversity, compared to core vaccine technology, is evident. On the other hand, antibiotic resistance is on the list of the top ten threats to global health. Immunomodulatory peptides that target a pathogen and modulate the immune system simultaneously are promising for the development of preventive and therapeutic molecules. Since investigating innate immunity in insects has led to prominent achievements in human immunology, such as toll-like receptor (TLR) discovery, we used the capacity of the immunomodulatory peptides of arthropods with concomitant antimicrobial or antitumor activity. An SVM-based machine learning classifier identified short immunomodulatory sequences encrypted in 643 antimicrobial peptides from 55 foe-to-friend arthropods. The critical features involved in efficacy and safety were calculated. Finally, 76 safe immunomodulators were identified. Then, molecular docking and simulation studies defined the target of the most optimal peptide ligands among all human cell-surface TLRs. SPalf2-453 from a crab is a cell-penetrating immunoadjuvant with antiviral properties. The peptide interacts with the TLR1/2 heterodimer. SBsib-711 from a blackfly is a TLR4/MD2 ligand used as a cancer vaccine immunoadjuvant. In addition, SBsib-711 binds CD47 and PD-L1 on tumor cells, which is applicable in cancer immunotherapy as a checkpoint inhibitor. MRh4-679 from a shrimp is a broad-spectrum or universal immunoadjuvant with a putative Th1/Th2-balanced response. We also implemented a pathway enrichment analysis to define fingerprints or immunological signatures for further in vitro and in vivo immunogenicity and reactogenicity measurements. Conclusively, combinatorial machine learning, molecular docking, and simulation studies, as well as systems biology, open a new opportunity for the discovery and development of multifunctional prophylactic and therapeutic lead peptides.
Irrational prescriptions, incorrect diagnoses, easy access, overuse, prophylaxis, and insufficient doses for antibiotic consumption have led to the development of drug-resistant microbial species [1,2]. Antibiotic resistance can be classified into intrinsic, acquired, and adaptive forms [3]. For example, the impermeability of the outer membrane of Gram-negative bacteria to large polar antibiotics or the lack of a target are intrinsic resistance mechanisms. Antibiotic inactivation, alteration in the target, activation of efflux pumps, and reduction of uptake are the main mechanisms of acquired antibiotic resistance. Microorganisms’ main adaptive antibiotic resistance is via the development of biofilm polymeric matrices and conversion to persister cells with slow growth [4,5]. Notably, the borders between these mechanisms are sometimes blurred, and a mechanism such as efflux pump activation can be intrinsic or acquired. Without adequate action, the world will face 10 million deaths annually due to antimicrobial resistance by 2050 [6].
On the other hand, current vaccination platforms have limitations in the provision of long-term protection, inadequate immunity in aged populations, and an inability to induce efficient cellular immunity [7]. Adjuvants are incorporated into the formulation of vaccines to boost the potency, spectrum, and durability of immune responses [8]. The underdevelopment of adjuvant discovery and diversity, compared to core vaccine technology, is evident [9]. To obtain approval for clinical use, adjuvants must meet four fundamental conditions. They should induce a potent cellular and humoral response and lead to long-term immunity. Regarding safety conditions, adjuvants should be nontoxic without causing autoimmune or allergic reactions [10]. The low diversity of vaccine adjuvants may originate from the fact that adjuvants are not developed for specific illness conditions, and a general adjuvant is incorporated in all formulations. From the viewpoint of pharmaceutical companies, the selection of an adjuvant for a new vaccine is a business decision, as the risk of using a new adjuvant may appear heavier than the benefit of a conventional adjuvant with a firm clinical track record [11]. So far, the FDA has approved fewer than ten adjuvants [12]. A timeline analysis shows that improved immunization by adding adjuvants to vaccine formulations has a history going back approximately one century, when Glenny et al., in 1926, precipitated antigens on alum particles [13,14]. Despite the strong induction of a humoral response, weak cellular immunity is provoked by aluminum salts. After, in 1997, squalene emulsion-based MF59 (oil-in-water) and adjuvant systems (AS) received approval for human vaccines. For example, monophosphoryl lipid A (MPL), a safe derivative of LPS, acts as a toll-like receptor 4 (TLR4) agonist in AS01, AS02, and AS04 adjuvants [9]. The COVID-19 pandemic has reignited the need to invest in strategic vaccine design and delivery approaches, leading to the licensing of lipid nanoparticles (LNPs). LNPs are delivery systems that can act as adjuvants [15]. However, delivery systems only promote antigen presentation by MHCs and do not affect the cytokine or other costimulatory signaling pathways.
With more than 80 FDA-approved peptide therapeutics, peptides are known as highly selective, specific, and biocompatible medications [16]. The capacity of immunomodulatory peptides with adjuvanticity properties (i.e., immunoadjuvants) has been extended recently in such a way that they not only boost the immune system but also display antimicrobial and antitumor activities themselves [17]. In the presence of immunomodulatory peptides that act as adjuvants, the maturation of a higher number of antigen-presenting cells (APCs), which elevates APC crosstalk with T-cells, is observed. This communication generates multifunctional T-cells and various classes of cytokines and antibodies [18]. In other words, adjuvants train adaptive immunity by stimulating innate immune cells and triggering pattern recognition receptor (PRR) signaling. These immunostimulatory adjuvants, such as pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), and TLR agonists, elevate antigen presentation on MHCs, induce costimulatory molecules, such as clusters of differentiation (CD) on APCs, and express secretion of cytokines [19]. The production of neutralizing antibodies and cytotoxic T-cells (CTLs) against defined antigens induces immune system memory cells for durable protection against infection during the vaccination process [9].
The development of broad-spectrum peptides as pan-antimicrobials and vaccine adjuvants has been reported previously. For example, the scope of granulocyte-colony stimulating factor (GCSF) activity ranges from vaccine adjuvant to antiviral immunotherapy [20]. Human defensins are host defense peptides (HDPs) or antimicrobial peptides (AMPs) that target pathogens and modulate the immune system concurrently, proposed as vaccine immunoadjuvants [21]. In parallel to modulating the immune system, AMPs disrupt membrane permeation and might interfere intracellularly with microbial transcription and translation processes [22]. AMPs, such as LL-37 derived from human cathelicidin, are HDPs with antimicrobial and immunomodulating properties linking innate and adaptive immunities [23]. A similar concept exists for developing cancer vaccine adjuvants with the extra ability to target tumor cells and which is applicable for cancer immunotherapy [24]. Safe and immunogenic MPL-adjuvanted vaccines are TLR4 agonists and have also been applied in cancer vaccines [25].
Although AMPs can induce the immune system, short immunomodulatory peptides are considered effective, safe, and economically feasible adjuvants for next-generation vaccine design [26]. Improvements in high throughput technologies, such as omics studies in parallel with machine learning and deep learning approaches, provide a rich pool of natural leads for drug discovery [27]. Invertebrates such as arthropods lack adaptive immunity and depend exclusively on innate immunity to defend themselves [28]. The slow rate of adjuvant discovery in humans originates from a strategy mainly based on adaptive immunity induction to trigger immunologic memory, ignoring other critical immunological elements required to boost vaccine efficacy. Favorably, considering innate immunity induction, which forms the adaptive immune reaction, has renovated the adjuvant’s mechanism of action [29].
Within this study, we investigated the most potent immunomodulating cryptic peptide fragments inside AMPs derived from arthropods using combinatorial machine learning, docking, simulation, and systems biology studies. We introduce immunoadjuvant peptides that target the pathogens or tumor cells as antimicrobial and anticancer agents in addition to their potency for inducing APCs and immune system stimulation. Because in vitro and in vivo adjuvanticity results are usually uncorrelated [11], we propose a so-called “systems adjuvantology” approach by identifying pathways and biological processes targeted by the identified adjuvants. These multifunctional peptides are a novel paradigm in peptide discovery and open a new trend in developing adjuvants and antibiotics.
A total of 643 AMPs of diverse arthropod species were retrieved from the InverPep database. Employing the VaxinPAD program, encrypted 10-mer and 15-mer peptides with immunomodulatory properties were extracted. Subsequently, immunomodulatory peptides with a score higher than 0.7 that were predicted to be nontoxic, nonallergenic, and nonhemolytic with no propensity to aggregation, known as “safe peptides”, were included for further analyses. The data reduction process identified 76 immunomodulatory peptides that met the efficacy and safety criteria.
To identify bifunctional peptides with concurrent properties in pathogen targeting and immune system modulation, MetaiAVP, AntiFP, and antiTBpred programs were used to define putative antiviral, antifungal, and antitubercular peptides with immunomodulatory properties out of the pool of 76 final candidates. Immunomodulatory peptides with anticancer characteristics were also determined. Finally, immunomodulators with the highest SVM score and the most potent antimicrobial or anticancer properties, possessing acceptable physiochemical characteristics, such as an appropriate isoelectric point (pI) and stability in an aqueous environment, were selected as the most optimal candidates to find the target TLR. The most potent immunomodulator with concomitant strong antitubercular, antibacterial, and antiangiogenic properties was named the universal adjuvant.
TLRs can be named the “Swiss Army” knife of immunity, prepared to react to numerous disease states. The most optimal immunomodulatory peptides that are reported in Table 2 were subjected to molecular docking analysis with all hTLRs, including TLR1/TLR2, TLR2, TLR2/TLR6, TLR4/MD2, TLR5, and TLR6, using the ClusPro 2.0 program. This aimed to identify the target with the most stable peptide–receptor complexes. The SPalf2-453 (HIRRRPKFRK) ligand, a decameric cationic antiviral peptide with strong immunomodulatory potency, was sourced from the antilipopolysaccharide factor (ALF-2), an AMP found in a mud crab Scylla paramamosain. Compared to a 35-mer antiviral peptide as the positive control, namely, An1a from spider (GFGCPLDQMQCHNHCQSVRYRGGYCTNFLKMTCKCY), SPalf2-453 displayed stronger immunomodulatory and comparable antiviral potency. While SPalf2-453 showed immunomodulatory and antiviral scores of about 1.04 and 0.998, respectively, An1a had immunomodulatory and antiviral scores of about 0.55 and 0.92. With a score of −1171.5, the TLR1/TLR2 heterodimer was the most optimal target for SPalf2-453 according to the ClusPro 2.0 program.
The LSsty1-174 ligand, a 10-mer peptide sequence “PCVQQPCPKC” derived from Litopenaeus stylirostris, a shrimp of the Penaeidae family, exhibits immunomodulatory and antifungal properties. With a score of −1408.8, TLR2 was the best target for LSsty1-174 using the ClusPro2.0 tool. The PPpp113-266 peptide (RVQERRFKRI), derived from PP113 AMP of an endoparasitic wasp (Pteromalus puparum), displays immunomodulatory and antitubercular properties.
