Pullenvalenes A-D: Nitric Oxide-Mediated Transcriptional Activation (NOMETA) Enables Discovery of Triterpene Aminoglycosides from Australian Termite Nest-Derived Fungi.

We report on the use of nitric oxide-mediated transcriptional activation (NOMETA) as an innovative means to detect and access new classes of microbial natural products encoded within silent biosynthetic gene clusters. A small library of termite nest- and mangrove-derived fungi and actinomyces was subjected to cultivation profiling using a miniaturized 24-well format approach (MATRIX) in the presence and absence of nitric oxide, with the resulting metabolomes subjected to comparative chemical analysis using UPLC-DAD and GNPS molecular networking. This strategy prompted study of Talaromyces sp. CMB-TN6F and Coccidiodes sp. CMB-TN39F, leading to discovery of the triterpene glycoside pullenvalenes A-D (1-4), featuring an unprecedented triterpene carbon skeleton and rare 6-O-methyl-N-acetyl-d-glucosaminyl glycoside residues. Structure elucidation of 1-4 was achieved by a combination of detailed spectroscopic analysis, chemical degradation, derivatization and synthesis, and biosynthetic considerations.

Australian Marine and Terrestrial Streptomyces-Derived Surugamides, and Synthetic Analogs, and Their Ability to Inhibit Dirofilaria immitis (Heartworm) Motility.

A bioassay-guided chemical investigation of a bacterium, Streptomyces sp. CMB-MRB032, isolated from sheep feces collected near Bathurst, Victoria, Australia, yielded the known polyketide antimycins A4a (1) and A2a (2) as potent inhibitors of Dirofilaria immitis (heartworm) microfilaria (mf) motility (EC50 0.0013-0.0021 µg/mL), along with the octapeptide surugamide A (3) and the new N-methylated analog surugamide K (4). With biological data suggesting surugamides may also exhibit activity against D. immitis, a GNPS molecular network analysis of a library of microbes sourced from geographically diverse Australian ecosystems identified a further five taxonomically and chemically distinct surugamide producers. Scaled-up cultivation of one such producer, Streptomyces sp. CMB-M0112 isolated from a marine sediment collected at Shorncliff, Qld, Australia, yielded 3 along with the new acyl-surugamides A1-A4 (5-8). Solid-phase peptide synthesis provided additional synthetic analogs, surugamides S1-S3 (9-11), while derivatization of 3 returned the semi-synthetic surugamide S4 (12) and acyl-surugamides AS1-AS3 (13-15). The natural acyl-surugamide A3 (7) and semi-synthetic acyl-surugamide AS3 (15) were shown to selectively inhibit D. immitis mf motility (EC50 3.3-3.4 µg/mL), however, unlike antimycins 1 and 2, were inactive against the gastrointestinal nematode Haemonchus contortus L1-L3 larvae (EC50 > 25 µg/mL) and were not cytotoxic to mammalian cells (human colorectal carcinoma SW620, IC50 > 30 µg/mL). A structure-activity relationship (SAR) study on the surugamides 3-15 revealed that selective acylation of the Lys3-ε-NH2 correlates with anthelmintic activity.

Activity Relationship of Poly(ethylenimine)-Based Liposomes as Group A Streptococcus Vaccine Delivery Systems.

Untreated group A Streptococcus (GAS) can lead to a range of life-threatening diseases, including rheumatic heart disease. To date, no therapeutic or prophylactic vaccines are commercially available to treat or prevent GAS infection. Development of a peptide-based subunit vaccine offers a promising solution, negating the safety issues of live-attenuated or inactive vaccines. Subunit vaccines administer small peptide fragments (antigens), which are typically poorly immunogenic. Therefore, these peptide antigens require formulation with an immune stimulant and/or vaccine delivery platform to improve their immunogenicity. We investigated polyelectrolyte complexes (PECs) and polymer-coated liposomes as self-adjuvanting delivery vehicles for a GAS B cell peptide epitope conjugated to a universal T-helper epitope and a synthetic toll-like receptor 2-targeting moiety lipid core peptide-1 (LCP-1). A structure-activity relationship of cationic PEC vaccines containing different external PEI-coatings (poly(ethylenimine); 10 kDa PEI, 25 kDa PEI, and a synthetic mannose-functionalized 25 kDa PEI) formed vaccines PEC-1, PEC-2, and PEC-3, respectively. All three PEC vaccines induced J8-specific systemic immunoglobulin G (IgG) antibodies when administered intranasally to female BALB/c mice without the use of additional adjuvants. Interestingly, PEC-3 induced the highest antibody titers among all tested vaccines, with the ability to effectively opsonize two clinically isolated GAS strains. A comparative study of PEC-2 and PEC-3 with liposome-based delivery systems was performed subcutaneously. LCP-1 was incorporated into a liposome formulation (DPPC, DPPG and cholesterol), and the liposomes were externally coated with PEI (25 kDa; Lip-2) or mannosylated PEI (25 kDa; Lip-3). All liposome vaccines induced stronger humoral immune responses compared to their PEC counterparts. Notably, sera of mice immunized with Lip-2 and Lip-3 produced significantly higher opsonic activity against clinically isolated GAS strains compared to the positive control, P25-J8 emulsified with the commercial adjuvant, complete Freund's adjuvant (CFA). This study highlights the capability of a PEI-liposome system to act as a self-adjuvanting vehicle for the delivery of GAS peptide antigens and protection against GAS infection.

Advances in Infectious Disease Vaccine Adjuvants.

Vaccines are one of the most significant medical interventions in the fight against infectious diseases. Since their discovery by Edward Jenner in 1796, vaccines have reduced the worldwide transmission to eradication levels of infectious diseases, including smallpox, diphtheria, hepatitis, malaria, and influenza. However, the complexity of developing safe and effective vaccines remains a barrier for combating many more infectious diseases. Immune stimulants (or adjuvants) are an indispensable factor in vaccine development, especially for inactivated and subunit-based vaccines due to their decreased immunogenicity compared to whole pathogen vaccines. Adjuvants are widely diverse in structure; however, their overall function in vaccine constructs is the same: to enhance and/or prolong an immunological response. The potential for adverse effects as a result of adjuvant use, though, must be acknowledged and carefully managed. Understanding the specific mechanisms of adjuvant efficacy and safety is a key prerequisite for adjuvant use in vaccination. Therefore, rigorous pre-clinical and clinical research into adjuvant development is essential. Overall, the incorporation of adjuvants allows for greater opportunities in advancing vaccine development and the importance of immune stimulants drives the emergence of novel and more effective adjuvants. This article highlights recent advances in vaccine adjuvant development and provides detailed data from pre-clinical and clinical studies specific to infectious diseases. Future perspectives into vaccine adjuvant development are also highlighted.

Development of Multilayer Nanoparticles for the Delivery of Peptide-Based Subunit Vaccine against Group A Streptococcus.

Peptide-based subunit vaccines include only minimal antigenic determinants, and, therefore, are less likely to induce allergic immune responses and adverse effects compared to traditional vaccines. However, peptides are weakly immunogenic and susceptible to enzymatic degradation when administered on their own. Hence, we designed polyelectrolyte complex (PEC)-based delivery systems to protect peptide antigens from degradation and improve immunogenicity. Lipopeptide (LCP-1) bearing J8 B-cell epitope derived from Group A Streptococcus (GAS) M-protein was selected as the model peptide antigen. In the pilot study, LCP-1 incorporated in alginate/cross-linked polyarginine-J8-based PEC induced high J8-specific IgG antibody titres. The PEC system was then further modified to improve its immune stimulating capability. Of the formulations tested, PEC-4, bearing LCP-1, alginate and cross-linked polylysine, induced the highest antibody titres in BALB/c mice following subcutaneous immunisation. The antibodies produced were more opsonic than those induced by mice immunised with other PECs, and as opsonic as those induced by antigen adjuvanted with powerful complete Freund's adjuvant.