Di-mannosylation enhances the adjuvant properties of adamantane-containing desmuramyl peptides in vivo.

Muramyl dipeptide (MDP) is the smallest essential peptidoglycan substructure capable of promoting both innate and adaptive immune responses. Herein, we report on the design, synthesis, and in vivo study of the adjuvant properties of two novel MDP analogs containing an achiral adamantyl moiety attached to the desmuramyl dipeptide (DMP) pharmacophore and additionally modified by one mannosyl subunit (derivative 7) or two mannosyl subunits (derivative 11). Mannose substructures were introduced in order to assess how the degree of mannosylation affects the immune response and nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) binding affinity, compared to the reference compound ManAdDMP. Both mannosylated MDP analogs showed improved immunomodulating properties, while the di-mannosylated derivative 11 displayed the highest, statistically significant increase in anti-OVA IgG production. In this study, for the first time, the di-mannosylated DMP derivative was synthesized and immunologically evaluated. Derivative 11 stimulates a Th-2-polarized type of immune reaction, similar to the reference compound ManAdDMP and MDP. Molecular dynamics (MD) simulations demonstrate that 11 has a higher NOD2 binding affinity than 7, indicating that introducing the second mannose significantly contributes to the binding affinity. Mannose interacts with key amino acid residues from the LRR hydrophobic pocket of the NOD2 receptor and loop 2.

Metal-Free Atom Transfer Radical Polymerization to Prepare Recylable Micro-Adjuvants for Dendritic Cell Vaccine.

In the development of dendritic cell (DC) vaccines, the maturation of DCs is a critical stage. Adjuvants play a pivotal role in the maturation of DCs, with a major concern being to ensure both efficacy and safety. This study introduces an innovative approach that combines high efficacy with safety through the synthesis of micro-adjuvants grafted with copolymers of 2-(methacrylamido) glucopyranose (MAG) and methacryloxyethyl trimethyl ammonium chloride (DMC). The utilization of metal-free surface-initiated atom transfer radical polymerization enables the production of safe and recyclable adjuvants. These micrometer-sized adjuvants surpass the optimal size range for cellular endocytosis, enabling the retrieval and reuse of them during the ex vivo maturation process, mitigating potential toxicity concerns associated with the endocytosis of non-metabolized nanoparticles. Additionally, the adjuvants exhibit a "micro-ligand-mediated maturation enhancement" effect for DC maturation. This effect is influenced by the shape of the particle, as evidenced by the distinct promotion effects of rod-like and spherical micro-adjuvants with comparable sizes. Furthermore, the porous structure of the adjuvants enables them to function as cargo-carrying "micro-shuttles", releasing antigens upon binding to DCs to facilitate efficient antigen delivery.

Trehalose diamide glycolipids augment antigen-specific antibody responses in a Mincle-dependent manner.

Many studies have investigated how trehalose glycolipid structures can be modified to improve their Macrophage inducible C-type lectin (Mincle)-mediated adjuvanticity. However, in all instances, the ester-linkage of α,ά-trehalose to the lipid of choice remained. We investigated how changing this ester-linkage to an amide influences Mincle signalling and agonist activity and demonstrated that Mincle tolerates this functional group change. In in vivo vaccination studies in murine and ovine model systems, using OVA or Mannheimia haemolytica and Mycoplasma ovipneumoniae as vaccine antigens, respectively, it was demonstrated that a representative trehalose diamide glycolipid was able to enhance antibody-specific immune responses. Notably, IgG titres against M. ovipneumoniae were significantly greater when using trehalose dibehenamide (A-TDB) compared to trehalose dibehenate (TDB). This is particularly important as infection with M. ovipneumoniae predisposes sheep to pneumonia.

Synthesis and immunostimulatory activity of sugar-conjugated TLR7 ligands.

Toll-like receptors (TLRs) are a type of pattern recognition receptors (PRRs), which are activated by recognizing pathogen-associated molecular patterns (PAMPs). The activation of TLRs initiates innate immune responses and subsequently leads to adaptive immune responses. TLR agonists are effective immuomodulators in vaccine adjuvants for infectious diseases and cancer immunotherapy. In exploring hydrophilic small molecules of TLR7 ligands using the cell-targeted property of a vaccine adjuvant, we conjugated 1V209, a small TLR7 ligand molecule, with various low or middle molecular weight sugar molecules that work as carriers. The sugar-conjugated 1V209 derivatives showed increased water solubility and higher immunostimulatory activity in both mouse and human cells compared to unmodified 1V209. The improved immunostimulatory potency of sugar-conjugates was attenuated by an inhibitor of endocytic process, cytochalasin D, suggesting that conjugation of sugar moieties may enhance the uptake of TLR7 ligand into the endosomal compartment. Collectively our results support that sugar-conjugated TLR7 ligands are applicable to novel drugs for cancer and vaccine therapy.

A CpG-riched plasmid as vaccine adjuvant reduce antigen dose of an inactivated Vibrio anguillarum vaccine in turbot (Scophthalmus maximus L.).

Inactivated vaccines are often applied with adjuvants in commercial fish farming. Although some mineral or non-mineral oil adjuvants show efficient improvement with inactivated vaccines, but sometimes bring side effects such as tissue adhesion and granulomatous lesion at the injection site. CpG ODN is a novel type of soluble adjuvant which has been proved to possess excellent advantages in fish vaccine development. In this study, we designed a tandem sequence of CpG ODN synthesized in plasmid pcDNA 3.1, and an inactivated Vibrio anguillarum vaccine developed in our previous work was chosen for determining the efficiency of the CpG-riched plasmids (pCpG) as an adjuvant. Results showed that pCpG we designed can offer higher immunoprotection with the vaccine. Interestingly, even below the minimum immune dosage of the vaccine, a high RPS of 84% was observed once the vaccine was administrated with the pCpG. Serum specific antibody titer, superoxide dismutase and total protein were enhanced and some immune genes related to both innate and adaptive immune response were upregulated, implying an effective auxiliary function of the pCpG. Totally, our study suggested that the pCpG is a potential and available adjuvant for turbot vaccine development.

Stereochemistry, lipid length and branching influences Mincle agonist activity of monoacylglycerides.

Herein, we report on the synthesis of a series of enantiomerically pure linear, iso-branched, and α-branched monoacyl glycerides (MAGs) in 63-72% overall yield. The ability of the MAGs to signal through human macrophage inducible C-type lectin (hMincle) using NFAT-GFP reporter cells was explored, as was the ability of the compounds to activate human monocytes. From these studies, MAGs with an acyl chain length ≥C22 were required for Mincle activation and the production of interleukin-8 (IL-8) by human monocytes. Moreover, the iso-branched MAGs led to a more pronounced immune response compared to linear MAGs, while an α-branched MAG containing a C-32 acyl chain activated cells to a higher degree than trehalose dibehenate (TDB), the prototypical Mincle agonist. Across the compound classes, the activity of the sn-1 substituted isomers was greater than the sn-3 counterparts. None of the representative compounds were cytotoxic, thus mitigating cytotoxicity as a potential mediator of cellular activity. Taken together, 6h (sn-1, iC26+1), 8a (sn-1, C32) and 8b (sn-3, C32) exhibited the best immunostimulatory properties and thus, have potential as vaccine adjuvants.

Synthesis and biological activities of amino acids functionalized α-GalCer analogues.

Invariant natural killer T-cells (iNKT-cells) are promising targets for manipulating the immune system, which can rapidly release a large amount of Th1 and Th2 cytokines upon the engagement of their T cell receptor with glycolipid antigens presented by CD1d. In this paper, we wish to report a novel series of α-GalCer analogues which were synthesized by incorporation of l-amino acid methyl esters in the C-6' position of glycolipid. The evaluation of these synthetic analogues for their capacities to stimulate iNKT-cells into producing Th1 and Th2 cytokines both in vitro and in vivo indicated that they were potent CD1d ligands and could stimulate murine spleen cells into a higher release of the Th1 cytokine IFN-γ in vitro. In vivo, Gly-α-GalCer (1) and Lys-α-GalCer (3) showed more Th1-biased responses than α-GalCer, especially analogue 3 showed the highest selectivity for IFN-γ production (IFN-γ/IL-4 = 5.32) compared with α-GalCer (IFN-γ/IL-4 = 2.5) in vivo. These novel α-GalCer analogues might be used as efficient X-ray crystallographic probes to reveal the relationship between glycolipids and CD1d proteins in α-GalCer/CD1d complexes and pave the way for developing new potent immunostimulating agents.

Enhanced Immunostimulatory Activity of CpG Oligodeoxynucleotide by the Combination of Mannose Modification and Incorporation into Nanostructured DNA.

The immunostimulatory activity of unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) could be improved via delivery to immune cells expressing Toll-like receptor 9 (TLR9). Previously, we showed that the polypod-like structured nucleic acid (polypodna), a nanostructured DNA comprised of three or more ODNs, was an efficient system for the delivery of CpG ODNs to immune cells. Because some TLR9-positive immune cells express mannose receptors (MR), the uptake of polypodna by immune cells can be further increased by its modification with mannose. In this study, we selected the phosphodiester CpG ODN, ODN1668, which has a sequence identical to CpG1668, and a hexapodna, a polypodna with six pods, to design a hexapodna that harbored ODN1668 or the mannosylated CpG ODN (Man-ODN1668) synthesized via modification of the 5'-terminal of ODN1668 with a synthesized mannose motif. By mixing ODN1668 or Man-ODN1668 with the hexapodna, ODN1668/hexapodna and Man-ODN1668/hexapodna were successfully formed with high yields. However, Man-ODN1668/hexapodna was found to induce a greater tumor necrosis factor-α release from TLR9- and MR-positive mouse peritoneal macrophages and macrophage-like J774.1 cells than Man-ODN1668 or ODN1668/hexapodna. These results indicate that the combination of mannose modification and incorporation into nanostructured DNA is a useful approach for enhancing the immunostimulatory activity of CpG ODN.

Optimized dose of synthetic analogues of Monophosphoryl lipid A as an effective alternative for formulating recombinant human papillomavirus vaccine.

Adjuvants are a crucial component of recombinant vaccines such as the human papillomavirus (HPV) vaccine. Monophosphoryl lipid A (MPL) extracted from Salmonella Minnesota lipopolysaccharide is used as an adjuvant for the HPV vaccine. Due to the limitations in accessibility and reproducibility of MPL, investigating synthetic analogues of MPL (synMPL) is urgently needed to overcome these limitations. In this study, female BALB/c mice were vaccinated by HPV vaccine formulated with synMPL and aluminum hydroxide gel in which the concentration of synMPL ranged from 0 to 100 µg/dose. Anti-HPV L1 VLP antibody was measured for each group through Indirect ELISA and compared with Cervarix and Gardasil vaccines as approved anti-HPV vaccines. SynMPL showed a concentration-dependent increase up to 50 µg/dose in the immunogenicity of the vaccine. Therefore, synMPL at concentration of 50 µg/dose was selected as optimum concentration. The GMT profiling of synMPL-formulated vaccine (named Papilloguard) and Cervarix was not statistically different (Mann-Whitney test). The Gardasil vaccine showed 10-fold lower GMT for anti-HPV 18 L1 VLP antibody but anti-HPV 16 L1 VLP antibody was similar to Cervarix and Papilloguard. The current findings suggest that the synMPL in combination with aluminum hydroxide could be used as a potential adjuvant candidate for human vaccine.

Preparation, Supramolecular Aggregation and Immunological Activity of the Bona Fide Vaccine Adjuvant Sulfavant S.

In aqueous conditions, amphiphilic bioactive molecules are able to form self-assembled colloidal structures modifying their biological activity. This behavior is generally neglected in preclinical studies, despite its impact on pharmacological development. In this regard, a significative example is represented by a new class of amphiphilic marine-inspired vaccine adjuvants, collectively named Sulfavants, based on the ß-sulfoquinovosyl-diacylglyceride skeleton. The family includes the lead product Sulfavant A (1) and two epimers, Sulfavant R (2) and Sulfavant S (3), differing only for the stereochemistry at C-2 of glycerol. The three compounds showed a significant difference in immunological potency, presumably correlated with change of the aggregates in water. Here, a new synthesis of diastereopure 3 was achieved, and the study of the immunomodulatory behavior of mixtures of 2/3 proved that the bizarre in vitro response to 1-3 effectively depends on the supramolecular aggregation states, likely affecting the bioavailability of agonists that can effectively interact with the cellular targets. The evidence obtained with the mixture of pure Sulfavant R (2) and Sulfavant S (3) proves, for the first time, that supramolecular organization of a mixture of active epimers in aqueous solution can bias evaluation of their biological and pharmacological potential.

One-Pot, Multicomponent Strategy for Designing Lymphoseek-Inspired Hetero-Glycoadjuvant@AuNPs.

Searching for vaccines (antigen and adjuvant) with easy preparation and strong T-cell response are crucial for antitumor immunity. In this work, to design lymphoseek-inspired vaccine possessing the abilities of promoting vaccine internalization and enhancing CD8+ T-cell responses, a simple multicomponent strategy is successfully utilized to fabricate lymph node and dendritic cell dual-targeting glycoadjuvant@AuNPs in one pot, where three different components, catechol-containing glycopolymer, HAuCl4 , and amine-terminal CpG (CpG-NH2 ) can react in a single step to generate target adjuvant. It is found that hetero-glycoadjuvant@AuNPs could increase adjuvant internalization and enhance the activation of bone-marrow-derived dendritic cells. Critically, lymphoseek-inspired vaccine potentiates antigen-specific CD8+ T-cell immune responses.

Immunostimulatory Phosphatidylmonogalactosyldiacylglycerols (PGDG) from the Marine Diatom Thalassiosira weissflogii: Inspiration for a Novel Synthetic Toll-Like Receptor 4 Agonist.

An unprecedented phosphatidylmonogalactosyldiacylglycerol pool (PGDG, 1) rich in polyunsaturated fatty acids was isolated from the marine diatoms Thalassiosira weissflogii. Here we report for the first time the NMR characterization of this rare lipid from marine organisms along with a synthetic strategy for the preparation of a PGDG analog (2). PGDG 1 exhibited immunostimulatory activity in human dendritic cells (DCs) and the synthetic PGDG 2 was prepared to explore its mechanism of action. A Toll-like receptor-4 (TLR-4) agonistic activity was evidenced in human and murine DCs underlying the antigen-specific T-cell activation of this class of molecules.

Synthetic Cancer-Targeting Innate Immune Stimulators Give Insights into Avidity Effects.

Multispecific and multivalent antibodies are seen as promising cancer therapeutics, and numerous antibody fragments and derivatives have been developed to exploit avidity effects that result in increased selectivity. Most of these multispecific and multivalent antibody strategies make use of recombinant expression of antigen-binding modules. In contrast, chemical synthesis and chemoselective ligations can be used to generate a variety of molecules with different numbers and combinations of binding moieties in a modular and homogeneous fashion. In this study we synthesized a series of targeted immune system engagers (ISErs) by using solid-phase peptide synthesis and chemoselective ligations. To explore avidity effects, we constructed molecules bearing different numbers and combinations of two "binder" peptides that target ephrin A2 and integrin α3 receptors and an "effector" peptide that binds to formyl peptide receptors and stimulates an immune response. We investigated various strategies for generating multivalent and multispecific targeted innate immune stimulators and studied their activities in terms of binding to cancer cells and stimulation of immune cells. This study gives insights into the influence that multivalency and receptor density have on avidity effects and is useful for the design of potential anticancer therapeutics.

Toll-like Receptor Agonist Conjugation: A Chemical Perspective.

Toll-like receptors (TLRs) are vital elements of the mammalian immune system that function by recognizing pathogen-associated molecular patterns (PAMPs), bridging innate and adaptive immunity. They have become a prominent therapeutic target for the treatment of infectious diseases, cancer, and allergies, with many TLR agonists currently in clinical trials or approved as immunostimulants. Numerous studies have shown that conjugation of TLR agonists to other molecules can beneficially influence their potency, toxicity, pharmacokinetics, or function. The functional properties of TLR agonist conjugates, however, are highly dependent on the ligation strategy employed. Here, we review the chemical structural requirements for effective functional TLR agonist conjugation. In addition, we provide similar analysis for those that have yet to be conjugated. Moreover, we discuss applications of covalent TLR agonist conjugation and their implications for clinical use.

Bioconjugation Approaches to Producing Subunit Vaccines Composed of Protein or Peptide Antigens and Covalently Attached Toll-Like Receptor Ligands.

Traditional vaccines derived from attenuated or inactivated pathogens are effective at inducing antibody-based protective immune responses but tend to be highly reactogenic, causing notable adverse effects. Vaccines with superior safety profiles can be produced by subunit approaches, utilizing molecularly defined antigens (e.g., proteins and polysaccharides). These antigens, however, often elicit poor immunological responses, necessitating the use of adjuvants. Immunostimulatory adjuvants have the capacity to activate antigen presenting cells directly through specific receptors (e.g., Toll-like receptors (TLRs)), resulting in enhanced presentation of antigens as well as the secretion of proinflammatory chemokines and cytokines. Consequently, innate immune responses are amplified and adaptive immunity is generated. Recently, site-specific conjugation of such immunostimulatory adjuvants (e.g., TLR ligands) onto defined antigens has shown superior efficacy over unconjugated mixtures, suggesting that the development of chemically characterized immunostimulatory adjuvants and optimized approaches for their conjugation with antigens may provide a better opportunity for the development of potent, novel vaccines. This review briefly summarizes various TLR agonists utilized as immunostimulatory adjuvants and focuses on the development of techniques (e.g., recombinant, synthetic, and semisynthetic) for generating adjuvant-antigen fusion vaccines incorporating peptide or protein antigens.

Site-specific effect of polar functional group-modification in lipids of TLR2 ligands for modulating the ligand immunostimulatory activity.

Toll-like receptor 2 (TLR2), a member of the TLR innate immune receptor family, recognizes lipoproteins from bacteria and modulates the immune response by inducing the expression of various cytokines. TLR2 has a large hydrophobic pocket that recognizes long fatty acyl groups on TLR2 ligands. However, few studies have focused on the property of the hydrophobic TLR2 pocket. Based on the X-ray crystal structure of TLR2, small polar regions were found in the hydrophobic TLR2 pocket. Interactions between the polar residues and ligands were explored here by designing and synthesizing a Pam2CSK4 derivative of the TLR2 ligands, containing an amide group within the lipid moiety. We evaluated the binding affinities and immunomodulatory activities of these ligands. Results suggested that the amide groups in the lipid chain interacted with the polar residues in the hydrophobic lipid-binding pocket of TLR2.

Nanoparticle vaccines against viral infections.

Despite numerous efforts, we still do not have prophylactic vaccines for many clinically relevant viruses, such as HIV, hepatitis C virus, Zika virus, and respiratory syncytial virus. Several factors have contributed to the current lack of effective vaccines, including the high rate of viral mutation, low immunogenicity of recombinant viral antigens, instability of viral antigenic proteins administered in vivo, sophisticated mechanisms of viral immune evasion, and inefficient induction of mucosal immunity by vaccine models studied to date. Some of these obstacles could be partially overcome by the use of vaccine adjuvants. Nanoparticles have been intensively investigated as vaccine adjuvants because they possess chemical and structural properties that improve immunogenicity. The use of nanotechnology in the construction of immunization systems has developed into the field of viral nanovaccinology. The purpose of this paper is to review and correlate recent discoveries concerning nanoparticles and specific properties that contribute to the immunogenicity of viral nanoparticle vaccines, bio-nano interaction, design of nanoparticle vaccines for clinically relevant viruses, and future prospects for viral nanoparticle vaccination.

Interaction between antimicrobial peptides and mycobacteria.

Mycobacteria can cause different severe health problems, including tuberculosis (TB). The treatment of TB with conventional antibiotics is successful, however, the number of multi-drug and extensively-drug resistant Mycobacterium tuberculosis strains increases. Moreover, many classical antimycobacterial antibiotics have severe side effects. Therefore, antimicrobial peptides (AMPs) seem to be good candidates for new therapeutic strategies. On the one hand AMPs can be used as a single drug or in combination with conventional antibiotics to directly kill mycobacteria, or on the other hand to act as immunstimulatory agents. This review summarizes the findings on the role of endogenous human AMPs being involved in TB, the antimycobacterial activity of various AMPs, and the molecular modes of action. Most active AMPs interact with the mycobacterial cell envelope and in particular with the mycomembrane and the plasma membrane. The mycomembrane is a very rigid membrane probably leading to a lower activity of the AMPs against mycobacteria as compared to other Gram-negative or Gram-positive bacteria. For some AMPs also other targets have been identified. Because of the complex environment of intracellular mycobacteria being trapped in the phagosome, within the macrophage, within the granuloma, within the lung, the external administration of AMPs in the latent phase of TB is a challenge. However, in the acute phase the AMPs can attack mycobacteria in a direct way. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Synthesis, Characterization and Immunological Evaluation of Self-Adjuvanting Group†A Streptococcal Vaccine Candidates Bearing Various Lipidic Adjuvanting Moieties.

Four group A streptococcal glycolipopeptide vaccine candidates with different lipidic adjuvanting moieties were prepared and characterized. The immunogenicity of the compounds was evaluated by macrophage and dendritic cell uptake studies and by in vivo quantification of systemic IgG antibody by ELISA. Three of the candidates showed significant induction of the IgG response.

Development of Improved Vaccine Adjuvants Based on the Saponin Natural Product QS-21 through Chemical Synthesis.

Vaccines based on molecular subunit antigens are increasingly being investigated due to their improved safety and more precise targeting compared to classical whole-pathogen vaccines. However, subunit vaccines are inherently less immunogenic; thus, coadministration of an adjuvant to increase the immunogenicity of the antigen is often necessary to elicit a potent immune response. QS-21, an immunostimulatory saponin natural product, has been used as an adjuvant in conjunction with various vaccines in numerous clinical trials, but suffers from several inherent liabilities, including scarcity, chemical instability, and dose-limiting toxicity. Moreover, little is known about its mechanism of action. Over a decade-long effort, beginning at the University of Illinois at Urbana-Champaign and continuing at the Memorial Sloan Kettering Cancer Center (MSKCC), the group of Prof. David Y. Gin accomplished the total synthesis of QS-21 and developed a practical semisynthetic approach to novel variants that overcome the liabilities of the natural product. First, semisynthetic QS-21 variants were designed with stable amide linkages in the acyl chain domain that exhibited comparable in vivo adjuvant activity and lower toxicity than the natural product. Further modifications in the acyl chain domain and truncation of the linear tetrasaccharide domain led to identification of a trisaccharide variant with a simple carboxylic acid side chain that retained potent adjuvant activity, albeit with reemergence of toxicity. Conversely, an acyl chain analogue terminating in a free amine was inactive but enabled chemoselective functionalization with radiolabeled and fluorescent tags, yielding adjuvant-active saponin probes that, unlike inactive congeners, accumulated in the lymph nodes in vaccinated mice and internalized into dendritic cells. Subtle variations in length, stereochemistry, and conformational flexibility around the central glycosidic linkage provided QS-21 variants with adjuvant activities that correlated with specific conformations found in molecular dynamics simulations. Notably, deletion of the entire branched trisaccharide domain afforded potent, truncated saponin variants with negligible toxicity and improved synthetic access, facilitating subsequent investigation of the triterpene core. The triterpene C4-aldehyde substituent, previously proposed to be important for QS-21 adjuvant activity, proved to be dispensable in these truncated saponin variants, while the presence of the C16 hydroxyl group enhanced activity. Novel adjuvant conjugates incorporating the small-molecule immunopotentiator tucaresol at the acyl chain terminus afforded adjuvant-active variants but without significant synergistic enhancement of activity. Finally, a new divergent synthetic approach was developed to provide versatile and streamlined access to additional linear oligosaccharide domain variants with modified sugars and regiochemistries, opening the door to the rapid generation of diverse, synthetically accessible analogues. In this Account, we summarize these multidisciplinary studies at the interface of chemistry, immunology, and medicine, which have provided critical information on the structure-activity relationships (SAR) of this Quillaja saponin class; access to novel, potent, nontoxic adjuvants for use in subunit vaccines; and a powerful platform for investigations into the mechanisms of saponin immunopotentiation.