Search

The unique mucosal immune system allows the generation of robust protective immune responses at the front line of pathogen encounters. The needle-free delivery route and cold chain-free logistic requirements also provide additional advantages in ease and economy. However, the development of mucosal vaccines faces several challenges, and only a handful of mucosal vaccines are currently licensed. These vaccines are all in the form of live attenuated or inactivated whole organisms, whereas no subunit-based mucosal vaccine is available. The selection of antigen, delivery vehicle, route and adjuvants for mucosal vaccination are highly important. This is particularly crucial for subunit vaccines, as they often fail to elicit strong immune responses. Emerging research is providing new insights into the biological and immunological uniqueness of mucosal tissues. However, many aspects of the mucosal immunology still await to be investigated. This article provides an overview of the current understanding of mucosal vaccination and discusses the remaining knowledge gaps. We emphasize that because of the potential benefits mucosal vaccines can bring from the biomedical, social and economic standpoints, the unmet goal to achieve mucosal vaccine success is worth the effort. [ABSTRACT FROM AUTHOR]

In the elderly, immunosenescence and malnourishment can contribute to increased risk and severity of upper respiratory tract infections (URTI). Gold kiwifruit (Actinidia chinensis 'Hort16') contains nutrients important for immune function and mitigation of symptoms of infection, including vitamins C and E, folate, polyphenols and carotenoids. The objective of the present study was to evaluate whether regular consumption of gold kiwifruit reduces symptoms of URTI in older people, and determine the effect it has on plasma antioxidants, and markers of oxidative stress, inflammation and immune function. A total of thirty-two community-dwelling people (≥65 years) participated in a randomised crossover study, consuming the equivalent of four kiwifruit or two bananas daily for 4 weeks, with treatments separated by a 4-week washout period. Participants completed the Wisconsin Upper Respiratory Symptom Survey-21 daily, and blood samples were collected at baseline and at the end of each treatment and washout period. Gold kiwifruit did not significantly reduce the overall incidence of URTI compared with banana, but significantly reduced the severity and duration of head congestion, and the duration of sore throat. Gold kiwifruit significantly increased plasma vitamin C, α-tocopherol and lutein/zeaxanthin concentrations, and erythrocyte folate concentrations, and significantly reduced plasma lipid peroxidation. No changes to innate immune function (natural killer cell activity, phagocytosis) or inflammation markers (high-sensitivity C-reactive protein, homocysteine) were detected. Consumption of gold kiwifruit enhanced the concentrations of several dietary plasma analytes, which may contribute to reduced duration and severity of selected URTI symptoms, offering a novel tool for reducing the burden of URTI in older individuals. [ABSTRACT FROM AUTHOR]

Kiwifruit is a good source of several vitamins and minerals and dietary fibre, and contains a number of phytochemicals; so kiwifruit potentially provides health benefits beyond basic nutrition. Consumption of green kiwifruit can have positive effects on cardiovascular health through antioxidant activity, inhibition of platelet aggregation and lowered TAG levels, and gut health through improving laxation, aiding digestion and promoting a healthy gut microflora. The importance of nutrition on immune function is well recognised, with deficiencies in vitamins A, C, E, B6 and B12, folic acid, Zn, Cu, Fe and Se being associated with impaired immune function and increased susceptibility to diseases. Evidence is growing that kiwifruit enhances immunity, with several small murine studies showing enhancement of innate and adaptive immune function. Few studies have examined the effect of kiwifruit on immune function in human subjects, but a recent study has revealed that kiwifruit up-regulates several ‘immune’ and ‘DNA and repair’-related gene sets, and down-regulates one gene set related to Ig secretion. Taken together, the evidence from the literature provides supporting data for designing a human intervention trial to validate the ability of kiwifruit to support immune function in healthy and immunocompromised populations. [ABSTRACT FROM PUBLISHER]

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-nucleotidaseA(S5nA).Arecombinant form of S5nA hydrolyzed AMP and ADP, but not ATP, to generate the immunomodulatory molecule adenosine. Michaelis-Menten kinetics revealed a Km of 169μMand a Vmax of 7550 nmol/mg/min for the substrate AMP. Furthermore, recombinant S5nA acted synergistically with S. pyogenes nuclease A to generate macrophagetoxic deoxyadenosine from DNA. The enzyme showed optimal activity betweenpH5 andpH6.5 and between 37 and 47 °C. 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 an S. pyogenes virulence factor. In conclusion, we have identified a novel S. pyogenes enzyme with 5-nucleotidase activity and immune evasion properties. [ABSTRACT FROM AUTHOR]

Group A Streptococcus (GAS) is a globally important pathogen causing a broad range of human diseases. GAS pili are elongated proteins with a backbone comprised repeating T-antigen subunits, which extend from the cell surface and have important roles in adhesion and establishing infection. No GAS vaccines are currently available, but T-antigen-based candidates are in pre-clinical development. This study investigated antibody-T-antigen interactions to gain molecular insight into functional antibody responses to GAS pili. Large, chimeric mouse/human Fab-phage libraries generated from mice vaccinated with the complete T18.1 pilus were screened against recombinant T18.1, a representative two-domain T-antigen. Of the two Fab identified for further characterization, one (designated E3) was cross-reactive and also recognized T3.2 and T13, while the other (H3) was type-specific reacting with only T18.1/T18.2 within a T-antigen panel representative of the major GAS T-types. The epitopes for the two Fab, determined by x-ray crystallography and peptide tiling, overlapped and mapped to the N-terminal region of the T18.1 N-domain. This region is predicted to be buried in the polymerized pilus by the C-domain of the next T-antigen subunit. However, flow cytometry and opsonophagocytic assays showed that these epitopes were accessible in the polymerized pilus at 37°C, though not at lower temperature. This suggests that there is motion within the pilus at physiological temperature, with structural analysis of a covalently linked T18.1 dimer indicating "knee-joint" like bending occurs between T-antigen subunits to expose this immunodominant region. This temperature dependent, mechanistic flexing provides new insight into how antibodies interact with T-antigens during infection.

Group A Streptococcus (GAS, Streptococcus pyogenes) is an exclusively human pathogen that causes a range of diseases, including pharyngitis, tonsillitis, impetigo, erysipelas, necrotizing fasciitis, and toxic shock syndrome. Post-streptococcal sequelae include acute rheumatic fever and rheumatic heart disease. The bacterium produces a large arsenal of virulence factors that contribute to host tissue adhesion/colonization, bacterial spread, and host immune evasion. Immune evasion factors include proteins that interfere with complement, a system of plasma proteins that are activated by pathogens resulting in a variety of reactions on the surface of the pathogen. This leads to the activation of active components with a variety of effector functions, such as cell lysis, opsonization, and chemotaxis of phagocytes to the site of infection. We have recently identified a novel "complement evasion factor" (CEF) in S. pyogenes. CEF directly interacts with complement proteins C1r, C1s, C3, and C5, interrupts all three complement pathways, and prevents opsonization of the bacterial surface with C3b. We here present methods used to analyze the complement interference of CEF., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Peptide vaccines offer an attractive strategy to induce highly specific immune responses while reducing potential side effects. However, peptides are often poorly immunogenic and unstable on their own, requiring the need for potentially toxic adjuvants or expensive chemical coupling. The novel peptide delivery platform PilVax utilizes the rigid pilus structure from Group A Streptococcus (GAS) to stabilize and amplify the peptide, and present it on the surface of the non-pathogenic food-grade bacterium Lactococcus lactis. Upon intranasal immunization, PilVax vaccines have proven to induce peptide-specific systemic and mucosal responses. PilVax provides an alternative method to develop mucosal vaccines that are inexpensive to produce and easy to administer., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The isolation of a single Group A Streptococcus (GAS) virulence determinant in functional investigations is challenging, as GAS employs a multitude of virulence factors. The redundancy between many surface proteins such as adhesins also adds complexity and difficulty. Lactococcus lactis is a non-pathogenic Gram-positive species related to GAS that can be an ideal surrogate organism to circumvent this problem. Genetic manipulation in L. lactis is easy, and the mechanisms for processing and cell wall-anchoring of surface proteins are similar to GAS. Lactococci have been extensively used to express heterologous surface proteins from other bacterial species, and modern molecular cloning tools and protocols have been developed. This chapter describes the workflow of generating recombinant L. lactis strains expressing GAS surface proteins and the validation and quantification of their surface expression.

The critical first step of Group A Streptococcus (GAS) pathogenesis is adhesion to the host pharyngeal and skin epithelial cell surfaces (Brouwer et al., FEBS Lett 590:3739-3757, 2016). Host-cell adhesion assays provide a straightforward model to study these host-pathogen interactions. Here, we describe the culturing of immortalized cell lines into monolayers to mimic host epithelia. Various GAS strains can then be added to study their adhesion properties. In addition, we describe the use of antibodies raised against the cell-surface components of GAS to study if these are able to neutralize the binding of GAS to the cell lines. This provides an indication if these cell-surface components are involved in adhesion and if antibodies generated against them function through neutralization.

Recently, the use of Galleria mellonella larvae as a nonmammalian model to simulate bacterial infectious diseases has shown to be a rapid, simple, and cost-effective alternative. The insect's innate immune response is remarkably similar to that of the vertebrates, and consists of both the cellular and the humoral immune response. Here, we provide a protocol for using G. mellonella larvae to study virulence of GAS, including the use of a health score system for quantitative analysis and the methods for assessing post-infection bacterial burden in vivo.

Bioluminescence is a rapid and cost-saving technology that enables monitoring of bacteria in real time in animal infection models. Here, we report a method for labeling GAS with a set of plasmids we have developed and deposited with Addgene. These plasmids can be used to either integrate firefly luciferase (Ffluc) or red-shifted firefly luciferase (FflucRT) into the GAS genome or to introduce the reporters on plasmids which have been stabilized with a toxin-antitoxin system to prevent loss of plasmid in the absence of antibiotic selection.

Group A Streptococcus (GAS) often exists as an asymptomatic colonizer of the upper respiratory tract in humans. Unsurprisingly, a high proportion of symptomatic infections caused by GAS include pharyngitis. While not usually life-threatening, these infections cause significant morbidity and economic burden/loss of productivity, and can have downstream life-threatening autoimmune consequences. Modeling asymptomatic colonization in animals is, therefore, a useful tool to dissect host-bacteria interactions and to evaluate efficacy of vaccines aimed at reducing the burden of carriage. Here we describe a mouse model of nasopharyngeal colonization using nasal challenge of susceptible mice and the evaluation of subsequent bacterial burden.

Group A Streptococcus (GAS) or Streptococcus pyogenes is responsible for an estimated 500,000 deaths worldwide each year. Protection against GAS infection is thought to be mediated by phagocytosis, enhanced by bacteria-specific antibody. There are no licenced GAS vaccines, despite many promising candidates in preclinical and early stage clinical development, the most advanced of which are based on the GAS M-protein. Vaccine progress has been hindered, in part, by the lack of a standardised functional assay suitable for vaccine evaluation. Current assays, developed over 50 years ago, rely on non-immune human whole blood as a source of neutrophils and complement. Variations in complement and neutrophil activity between donors result in variable data that is difficult to interpret. We have developed an opsonophagocytic killing assay (OPKA) for GAS that utilises dimethylformamide (DMF)-differentiated human promyelocytic leukemia cells (HL-60) as a source of neutrophils and baby rabbit complement, thus removing the major sources of variation in current assays. We have standardised the OPKA for several clinically relevant GAS strain types (emm1, emm6 and emm12) and have shown antibody-specific killing for each emm-type using M-protein specific rabbit antisera. Specificity was demonstrated by pre-incubation of the antisera with homologous M-protein antigens that blocked antibody-specific killing. Additional qualifications of the GAS OPKA, including the assessment of the accuracy, precision, linearity and the lower limit of quantification, were also performed. This GAS OPKA assay has the potential to provide a robust and reproducible platform to accelerate GAS vaccine development., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The lack of standardised protocols for the assessment of functional antibodies has hindered Streptococcus pyogenes research and the development of vaccines. A robust, high throughput opsonophagocytic bactericidal assay to determine protective antibodies in human and rabbit serum has been developed that utilises bioluminescence as a rapid read out., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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 169 μm and a Vmax of 7550 nmol/mg/min for the substrate AMP. Furthermore, recombinant S5nA acted synergistically with S. pyogenes nuclease A to generate macrophage-toxic deoxyadenosine from DNA. The enzyme showed optimal activity between pH 5 and pH 6.5 and between 37 and 47 °C. Like other 5'-nucleotidases, S5nA requires divalent cations and was active in the presence of Mg(2+), Ca(2+), or Mn(2+). However, Zn(2+) 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 an S. pyogenes virulence factor. In conclusion, we have identified a novel S. pyogenes enzyme with 5'-nucleotidase activity and immune evasion properties., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Group A streptococcus is a strict human pathogen that can cause a wide range of diseases, such as tonsillitis, impetigo, necrotizing fasciitis, toxic shock, and acute rheumatic fever. Modeling human diseases in animals is complicated, and rapid, simple, and cost-effective in vivo models of GAS infection are clearly lacking. Recently, the use of non-mammalian models to model human disease is starting to re-attract attention. Galleria mellonella larvae, also known as wax worms, have been investigated for modeling a number of bacterial pathogens, and have been shown to be a useful model to study pathogenesis of the M3 serotype of GAS. In this study we provide further evidence of the validity of the wax worm model by testing different GAS M-types, as well as investigating the effect of bacterial growth phase and incubation temperature on GAS virulence in this model. In contrast to previous studies, we show that the M-protein, among others, is an important virulence factor that can be effectively modeled in the wax worm. We also highlight the need for a more in-depth investigation of the effects of experimental design and wax worm supply before we can properly vindicate the wax worm model for studying GAS pathogenesis.