Galleria mellonella (greater wax moth or honeycomb moth) has been introduced as an alternative model to study microbial infections. G. mellonella larvae can be easily and inexpensively obtained in large numbers and are simple to use as they don't require special lab equipment. There are no ethical constraints and their short life cycle makes them ideal for large-scale studies. Although insects lack an adaptive immune response, their innate immune response shows remarkable similarities with the immune response in vertebrates. This review gives a current update of what is known about the immune system of G. mellonella and provides an extensive overview of how G. mellonella is used to study the virulence of Gram-positive and Gram-negative bacteria. In addition, the use of G. mellonella to evaluate the efficacy of antimicrobial agents and experimental phage therapy are also discussed. The review concludes with a critical assessment of the current limitatons of G. mellonella infection models.

Peptide- and protein-based therapeutics offer realized and potential benefits to health, due to their potent bioactivity, high specificity, and favorable safety characteristics. However, their widespread clinical application is constrained by inherent limitations, including rapid enzymatic degradation, poor membrane permeability, and a reliance on parenteral administration, which reduces patient adherence. To overcome these challenges, extensive research has explored non-invasive delivery strategies, including topical, transdermal, and oral formulations. Despite promising advances in these delivery strategies, they are yet to overcome substantial biological and physicochemical barriers in peptide and protein therapeutics, such as enzymatic degradation in the gastrointestinal tract, limited epithelial transport, and inherently low systemic bioavailability. This review provides a comprehensive and up-to-date analysis of the structural and physiological barriers influencing peptide and protein bioavailability and therapeutic efficacy. It critically examines key challenges associated with various administration routes, including topical, transdermal, oral (including delivery targeting the brain), and others. Furthermore, it explores innovative strategies to enhance peptide and protein stability and bioavailability, including chemical modifications, enzyme inhibitors, penetration enhancers, physical delivery technologies, and advanced nanoparticulate formulations. Additionally, emerging trends in formulation optimization, regulatory considerations, and translational pathways for clinical implementation are discussed. By addressing these critical challenges and highlighting recent advances, this review serves as a roadmap for the development of next-generation peptide and protein therapeutics with improved stability and efficacy, and enhanced patient adherence, which is needed to fully realize the true potential of this class of therapeutics.

Streptococcus pyogenes is an important human pathogen that causes a wide range of diseases. Using bioinformatics analysis of the complete S. pyogenes strain SF370 genome, we have identified a novel S. pyogenes virulence factor, which we termed streptococcal 5-nucleotidase A (S5nA). A recombinant form of S5nA hydrolyzed AMP and ADP, but not ATP, to generate the immunomodulatory molecule adenosine. Michaelis-Menten kinetics revealed a Km of 9.3 nmol and a Vmax of 7550 nmol/mg/min for the substrate AMP. Furthermore, rS5nA acted synergistically with S. pyogenes nuclease A (SpnA) to generate macrophage-toxic deoxyadenosine from DNA. The enzyme showed optimal activity between pH5 and pH6.5, and between 37C and 47C. Like other 5-nucleotidases, S5nA requires divalent cations and was active in the presence of Mg2+, Ca2+ or Mn2+. However, Zn2+ inhibited the enzymatic activity. Structural modeling combined with mutational analysis revealed a highly conserved catalytic dyad, as well as conserved substrate and cation-binding sites. Recombinant S5nA significantly increased the survival of the non-pathogenic bacterium Lactococcus lactis during a human whole blood killing assay in a dose-dependent manner suggesting a role as a S. pyogenes virulence factor. In conclusion, we have identified a novel S. pyogenes enzyme with 5-nucleotidase activity and immune evasion properties.

Pili of Group A Streptococcus (GAS) are surface-exposed structures involved in adhesion and colonisation of the host during infection. The major protein component of the GAS pilus is the T-antigen, which multimerises to form the pilus shaft. There are currently no licenced vaccines against GAS infections and the T-antigen represents an attractive target for vaccination. We have generated a multivalent vaccine called TeeVax1, a recombinant protein that consists of a fusion of six T-antigen domains. Vaccination with TeeVax1 produces opsonophagocytic antibodies in rabbits and confers protective efficacy in mice against invasive disease. Two further recombinant proteins, TeeVax2 and TeeVax3 were constructed to cover 12 additional T-antigens. Combining TeeVax1–3 produced a robust antibody response in rabbits that was cross-reactive to a full panel of 21 T-antigens, expected to provide over 95% vaccine coverage. These results demonstrate the potential for a T-antigen-based vaccine to prevent GAS infections.

The human pathogen Group A Streptococcus (GAS) produces pili that are involved in adhesion and colonisation of the host. These surface-exposed pili are immunogenic and therefore represent an attractive target for vaccine development. The pilus is encoded in the genomic region known as the fibronectin-collagen-T-antigen (FCT)-region, of which at least nine different types have been identified. In this study we investigate expressing two of the most common FCT-types (FCT-3 and FCT-4) in the food-grade bacteria Lactococcus lactis for use as a mucosal vaccine. We show that mucosally delivered L. lactis expressing GAS pili generates specific antibody responses in rabbits. Rabbit anti-pilus antibodies were shown to have both a neutralising effect on bacterial adhesion, and immunised rabbit antiserum was able to facilitate immune-mediated killing of bacteria via opsonophagocytosis. Furthermore, intranasal immunisation of mice improved clearance rates of GAS after nasopharyngeal challenge. These results demonstrate the potential for a novel, pilus-based vaccine to protect against GAS infections.