Synthesis and Evaluation of a Self-Adjuvanting Pseudomonal Vaccine Based on Major Outer Membrane Porin OprF Epitopes Formulated with Low-Toxicity QS-21-Containing Liposomes.

Pseudomonas aeruginosa (PA) is a Gram-negative pathogen that the World Health Organization has ranked as a priority 1 (critical) threat. One potential prophylactic approach to preventing or reducing the incidence of PA would be development of a long sought-after vaccine. Both antibody and CD4+ T-cell responses have been noted as playing key roles in protection against infection. In these studies, we have designed a prototype vaccine consisting of several known linear B-cell epitopes derived from an outer membrane porin F (OprF). The resulting thiol-containing protein was conjugated to a version of the lipopeptide-based Toll-like receptor agonist Pam3CysSK4Mal (10) containing a maleimide moiety and formulated into dipalmitoylphosphatidylcholine (DPPC)/cholesterol (Chol) liposomes. Mice immunized with the resulting vaccine generated antibodies that bound PA14 (serotype O10) in vitro and induced opsonization in the presence of rabbit complement and murine macrophage RAW264.7 cells. The liposome was optimized to contain 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG), Chol, Pam3CysSK4-OprF (12) and the Quillaja saponaria-derived saponin adjuvant QS-21. The resulting vaccine formulation produced significantly higher antibody titers, increased the IgG2a antibody isotype, and increased the number of IgG-producing B-cells as well as splenic primed T-cells. In summary, the liposomal vaccine platform was found highly useful for the generation of a robust and balanced TH1/TH2 response.

Synthesis and Immunological Evaluation of a Single Molecular Construct MUC1 Vaccine Containing l-Rhamnose Repeating Units.

A rhamnose targeting strategy for generating effective anticancer vaccines was successful in our previous studies. We showed that by utilizing natural anti-rhamnose antibodies, a rhamnose-containing vaccine can be targeted to antigen-presenting cells, such as dendritic cells. In this case, rhamnose (Rha) was linked directly to the liposomes bearing the antigen. However, in the current approach, we conjugated a multivalent Tri-Rha ligand with the antigen itself, making it a single component vaccine construct, unlike the previous two-component vaccine construct where Rha cholesterol and Mucin1 (MUC1) antigen were both linked separately to the liposomes. Synthesis required the development of a linker for coupling of the Rha-Ser residues. We compared those two systems in a mouse model and found increased production of anti-MUC1 antibodies and more primed antigen-specific CD4+ T cells in both of the targeted approaches when compared to the control group, suggesting that this one-component vaccine construct could be a potential design used in our MUC1 targeting mechanisms.

γ-Secretase inhibitors and modulators: Mechanistic insights into the function and regulation of γ-Secretase.

Over two decades, γ-secretase has been the target for extensive therapeutic development due to its pivotal role in pathogenesis of Alzheimer's disease and cancer. However, it has proven to be a challenging task owing to its large set of substrates and our limited understanding of the enzyme's structural and mechanistic features. The scientific community is taking bigger strides towards solving this puzzle with recent advancement in techniques like cryogenic electron microscopy (cryo-EM) and photo-affinity labelling (PAL). This review highlights the significance of the PAL technique with multiple examples of photo-probes developed from γ-secretase inhibitors and modulators. The binding of these probes into active and/or allosteric sites of the enzyme has provided crucial information on the γ-secretase complex and improved our mechanistic understanding of this protease. Combining the knowledge of function and regulation of γ-secretase will be a decisive factor in developing novel γ-secretase modulators and biological therapeutics.

Biochemical and microbiological evaluation of N-aryl urea derivatives against mycobacteria and mycobacterial hydrolases.

A focused library of 24 N-aryl urea derivatives was prepared and evaluated against serine esterases of Mycobacterium tuberculosis (Mtb) Rv3802c and Mtb Ag85C. The members of the library were evaluated for both selectivity and mode of inhibition. Furan-based urea derivative 6c was found to be the most potent non-covalent inhibitor of Rv3802c with a K i value of 5.2 ± 0.7 µM. On the other hand, triazole-based ureas 10a and 10b selectively inhibited Ag85C irreversibly with a k inact/K i value of 2.3 ± 0.3 and 5.5 ± 0.4 × 10-3 µM-1 min-1, respectively. The library was also evaluated for minimum inhibitory concentration (MIC) against two strains of Mtb, Mycobacterium smegmatis, and Mycobacterium abscessus. Compounds 4a and 4c were active against Mtb H37Rv mc26206 with MIC values of 3.12 and 1.5 µM, respectively. Closely related 4e showed similar activity against Mtb H37Rv mc26206 but also possessed activity against Mtb H37Ra, Mycobacterium smegmatis and Mycobacterium abscessus. Compounds 4a, 4c, and 4e all contained a common 1-(cyclohexylmethyl)-3-phenylurea motif. In summary, we identified a selective non-covalent inhibitor of Rv3802c and covalently irreversible inhibitors of Ag85C as well as the 1-(cyclohexylmethyl)-3-phenylurea motif which showed activity against a variety of mycobacteria.

Development of a Bioconjugate Platform for Modifying the Immune Response of Autoreactive Cytotoxic T Lymphocytes Involved in Type 1 Diabetes.

Type 1 diabetes (T1D) is an autoimmune disorder characterized by autoimmune cell mediated destruction of pancreatic beta cells. Pancreatic beta cells are the only source of insulin in the body. T1D patients then have to depend on insulin injections for their lifetime. Insulin injection can modulate the blood sugar levels, but insulin has little effect on the autoimmune process. Altered peptide ligands (APL) derived from known autoantigens in T1D are able to induce tolerance in autoreactive cells in T1D animal models, but are currently unable to elicit this protection in humans. There is a need to improve immunogenicity of the APLs, as these short peptides can be easily degraded by enzymes in the blood. GAD546-554 is a dominant epitope recognized by autoreactive T cells in the nonobese diabetic (NOD) mouse model that can cause destruction of beta cells. Alanine substitution at the eighth position of GAD546-554 peptide (APL9) induced tolerance in a GAD546-554 specific cytotoxic T lymphocyte clone. To improve the antigen presentation and endosomal escape of APL9, we developed a bioconjugate platform that consists of a liposome containing a bioconjugate of APL9 and toll-like receptor 2 ligand Pam3CysSK4 as well as an antibody against macrophage protein F4/80. APL9 bioconjugate liposome with F4/80 antibody was able to induce tolerance in a GAD 546-554 specific clone. Diabetic NOD splenocytes pretreated with APL9 bioconjugate were also not able to transfer diabetes into prediabetic NOD recipient mice. This work is beneficial to prevent T1D as an immunotherapy strategy to render autoreactive immune cells more tolerant of beta cells.

Liposomal Fc Domain Conjugated to a Cancer Vaccine Enhances Both Humoral and Cellular Immunity.

Targeted delivery of antigens to antigen-presenting cells (APCs) by utilizing natural anticarbohydrate antibodies is a promising approach for selective uptake and enhanced antigen presentation. Previously, we reported that in the presence of a natural antibody, anti-rhamnose antibody (anti-Rha), the bacterial sugar rhamnose conjugated with liposomal cancer antigen MUC1-Tn enhances antigen presentation by APCs such as dendritic cells by targeting Fc gamma receptors. The idea was to utilize the natural human anti-Rha antibodies present in human serum for targeted delivery of cancer-specific antigens. Recently, we found that the IgG3 antibody isotype was the most prevalent anti-Rha antibody generated in mice immunized with rhamnose-Ficoll (Rha-Ficoll) antigen. In this manuscript, we have conjugated the murine IgG3-Fc with a MUC1-containing cancer vaccine and compared the humoral and cellular immune response to this vaccine with one targeted via the human anti-Rha antibody and to the MUC1 vaccine alone. This Fc approach enhanced antibody production and T-cell proliferation almost to the same level as using the anti-Rha antibody. These results suggest that targeting Fc directly to dendritic cells can be an alternative approach to human anti-Rha for generating effective antigen-primed T-cells.

Augmenting Vaccine Immunogenicity through the Use of Natural Human Anti-rhamnose Antibodies.

Utilizing natural antibodies to augment vaccine immunogenicity is a promising approach toward cancer immunotherapy. Anti-rhamnose (anti-Rha) antibodies are some of the most common natural anti-carbohydrate antibodies present in human serum. Therefore, rhamnose can be utilized as a targeting moiety for a rhamnose-containing vaccine to prepare an effective vaccine formulation. It was shown previously that anti-Rha antibody generated in mice binds effectively with Rha-conjugated vaccine and is picked up by antigen presenting cells (APCs) through stimulatory Fc receptors. This leads to the effective uptake and processing of antigen and eventually presentation by major histocompatibility complex (MHC) molecules. In this article, we show that natural human anti-Rha antibodies can also be used in a similar mechanism and immunogenicity can be enhanced by targeting Rha-conjugated antigens. In doing so, we have purified human anti-Rha antibodies from human serum using a rhamnose affinity column. In vitro, human anti-Rha antibodies are shown to enhance the uptake of a model antigen, Rha-ovalbumin (Rha-Ova), by APCs. In vivo, they improved the priming of CD4+ T cells to Rha-Ova in comparison to non-anti-Rha human antibodies. Additionally, increased priming of both CD4+ and CD8+ T cells toward the cancer antigen MUC1-Tn was observed in mice that received human anti-Rha antibodies prior to vaccination with a rhamnose-modified MUC1-Tn cancer vaccine. The vaccine conjugate contained Pam3CysSK4, a Toll-like receptor (TLR) agonist linked via copper-free cycloaddition chemistry to a 20-amino-acid glycopeptide derived from the tumor marker MUC-1 containing the tumor-associated carbohydrate antigen α- N-acetyl galactosamine (GalNAc). The primed CD8+ T cells released IFN-γ and killed tumor cells. Therefore, we have confirmed that human anti-Rha antibodies can be effectively utilized as a targeting moiety for making an effective vaccine.

Synthesis of Oligosaccharide Components of the Outer Core Domain of P. aeruginosa Lipopolysaccharide Using a Multifunctional Hydroquinone-Derived Reducing-End Capping Group.

The synthesis of a trisaccharide (common to glycoform I and II) and a tetrasaccharide (common to glycoform I) from the outer core domain of Pseudomonas aeruginosa lipopolysaccharide (LPS) using a novel hydroquinone-based reducing-end capping group is reported. This multifunctional capping group was utilized as purification handle and was stable toward many common transformations in oligosaccharide synthesis. The access to outer-core LPS antigens with a TBDPS-protected hydroquinone (TPH) at the reducing end will be useful for glycan array and therapeutic glycoconjugate synthesis.

Oligosaccharide synthesis on soluble high-molecular weight pHEMA using a photo-cleavable linker.

Oligosaccharide synthesis on organic solvent soluble, high molecular weight poly(2-hydroxyethylmethylacrylate) (pHEMA) is described. The pHEMA-bound oligosaccharide could be recovered after each reaction in 90-95% yield using a precipitation method. The methodology was used to synthesize a model tri-galactoside in 48% overall yield and a trisaccharide from the outer core domain of Pseudomonas aeruginosa lipopolysacchride (LPS) in 39% yield. The use of a photo-cleavable linker is also demonstrated to produce reducing-end protected oligosaccharides.

Recent Advances in Subunit Vaccine Carriers.

The lower immunogenicity of synthetic subunit antigens, compared to live attenuated vaccines, is being addressed with improved vaccine carriers. Recent reports indicate that the physio-chemical properties of these carriers can be altered to achieve optimal antigen presentation, endosomal escape, particle bio-distribution, and cellular trafficking. The carriers can be modified with various antigens and ligands for dendritic cells targeting. They can also be modified with adjuvants, either covalently or entrapped in the matrix, to improve cellular and humoral immune responses against the antigen. As a result, these multi-functional carrier systems are being explored for use in active immunotherapy against cancer and infectious diseases. Advancing technology, improved analytical methods, and use of computational methodology have also contributed to the development of subunit vaccine carriers. This review details recent breakthroughs in the design of nano-particulate vaccine carriers, including liposomes, polymeric nanoparticles, and inorganic nanoparticles.