Mucosal vaccination: onward and upward.

INTRODUCTION: 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. AREAS COVERED: 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. EXPERT OPINION: 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.

Naturally acquired functional antibody responses to group A Streptococcus differ between major strain types.

Group A Streptococcus (GAS) is a leading human pathogen for which there is no licensed vaccine. Infections are most common in young children and the elderly suggesting immunity accumulates with exposure until immune senescence in older age. Though protection has been postulated to be strain type specific, based on the M-protein (emm-type), the antigenic basis of population-level immunity remains poorly understood. Naturally acquired GAS antibody responses were investigated using intravenous immunoglobulin (IVIG), which contains pooled immunoglobulins from thousands of healthy human donors, as a surrogate for population immunity. Functional opsonophagocytic killing assays were conducted with GAS strains (n = 6) representing the three major emm-pattern types (emm12, A-C pattern; emm53, D-pattern; and emm75, E-pattern). While IVIG induced opsonophagocytic killing of all GAS strains tested, specificity assays showed the profile of protective antibodies differed considerably between emm-types. Antibodies targeting the M-protein were a major component of the functional IVIG antibody response for emm12 and emm53 strains but not for emm75 strains. The striking differences in the contribution of M-protein specific antibodies to killing suggest naturally acquired immunity differs between strains from the major emm-patterns. This challenges the dogma that M-protein is the primary protective antigen across all GAS straintypes. IMPORTANCE Group A Streptococcus (GAS) is a globally important pathogen. With the surge of invasive GAS infections that have occurred in multiple countries, contemporaneous with the relaxation of COVID-19 pandemic restrictions, there is increased interest in the mechanisms underpinning GAS immunity. We utilized intravenous immunoglobulin (IVIG), pooled immunoglobulins from thousands of healthy donors, as a surrogate for population-level immunity to GAS, and explored the contribution of strain-specific (M-type specific) antibodies to GAS immunity using functional killing assays. This revealed striking differences between major strain types as to the contribution of strain specific antibodies to killing. For GAS strains belonging to the E pattern group, M-type specific antibodies do not mediate killing and immunity, which contrasts with strains belonging to pattern A-C and D groups. This challenges the historical dogma, originally proposed by Rebecca Lancefield in the 1950-1960s, that the M-protein is the major protective antigen across all GAS strain types.

Methods to Analyze the Contribution of Complement Evasion Factor (CEF) to Streptococcus pyogenes Virulence.

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.

Identification of an immunodominant region on a group A Streptococcus T-antigen reveals temperature-dependent motion in pili.

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.

The effects of sugar in drinking water on Streptococcus pyogenes colonisation in a murine nasopharyngeal infection model.

The number of sugar-sweetened beverages consumed per day has been associated with an increased risk of acute rheumatic fever, an autoimmune disease triggered by superficial Streptococcus pyogenes infection. To explore if there could be a biological basis for this association, we used a mouse model of S. pyogenes nasopharyngeal colonisation combined with a dietary intervention. We observed an increased bacterial load in the nasopharynx of mice receiving sucrose drinking water post-infection, suggesting that high sucrose intake promotes S. pyogenes growth and/or survival. This provides new insight into the potential biological basis behind the association seen in humans.

Complement evasion factor (CEF), a novel immune evasion factor of Streptococcus pyogenes.

Streptococcus pyogenes, a leading human pathogen, is responsible for a wide range of diseases, including skin and soft tissue infections and severe invasive diseases. S. pyogenes produces a large arsenal of virulence factors, including several immune evasion factors. We have identified an open reading frame (spy0136) in the S. pyogenes SF370 genome encoding a protein of unknown function. Using recombinant Spy0136 in a pull-down assay with human plasma and ELISA, we have identified four complement proteins (C1r, C1s, C3, and C5) as binding partners. Treatment of the complement proteins with PNGase F abrogated binding to C1s, C3, and C5, indicating glycan-dependent interactions. rSpy0136 inhibited complement-mediated hemolysis and interfered with all three complement pathways in a Wieslab complement assay. Furthermore, rSpy0136 inhibited deposition of the C3b opsonin and the membrane attack complex (MAC) on the surface of S. pyogenes. We therefore named the previously unknown protein 'complement evasion factor' (CEF).An S. pyogenes Δspy0136/cef deletion mutant showed decreased virulence in an in-vitro whole blood killing assay and a Galleria mellonella (wax moth) infection model. Furthermore, an L. lactis spy0136/cef gain-of-function mutant showed increased survival during growth in whole human blood. Analysis of serum samples from patients with invasive S. pyogenes revealed Spy0136/CEF sero-conversion indicating expression during disease. In summary, we have identified a novel S. pyogenes immune evasion factor that binds to several complement proteins to interfere with complement function. This is the first example of a S. pyogenes virulence factor binding to several different target proteins via glycan-dependent interactions.

PilVax: A Novel Platform for the Development of Mucosal Vaccines.

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.

A multivalent T-antigen-based vaccine for Group A Streptococcus.

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.

Generation of Bioluminescent Group A Streptococcus for Biophotonic Imaging.

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.

Using Lactococcus lactis as Surrogate Organism to Study Group A Streptococcus Surface Proteins.

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.

Assays to Analyze Adhesion of Group A Streptococcus to Host Cells.

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.

The Use of Galleria mellonella (Wax Moth) as an Infection Model for Group A Streptococcus.

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.

A Mouse Nasopharyngeal Colonization Model for Group A Streptococcus.

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.

Cell wall-anchored 5'-nucleotidases in Gram-positive cocci.

5'-nucleotidases (5'-NTs) are enzymes that catalyze the hydrolysis of nucleoside monophosphates to produce nucleosides and phosphate. Since the identification of adenosine synthase A (AdsA) in Staphylococcus aureus in 2009, several other 5'-NTs have been discovered in Gram-positive cocci, mainly in streptococci. Despite some differences in substrate specificity, pH range and metal ion requirements, all characterized 5'-NTs use AMP and ADP, and in some cases ATP, to produce the immunosuppressive adenosine, which dampens pro-inflammatory immune responses. Several 5'-NTs are also able to use dAMP as substrate to generate deoxy-adenosine which is cytotoxic for macrophages. A synergy between 5'-NTs and exonucleases which are commonly expressed in Gram-positive cocci has been described, where the nucleases provide dAMP as a cleavage product from DNA. Some of these nucleases produce dAMP by degrading the DNA backbone of neutrophil extracellular traps (NETs) resulting in a "double hit" strategy of immune evasion. This Micro Review provides an overview of the biochemical properties of Gram-positive cell wall-anchored 5'-NTs and their role as virulence factors. A potential use of 5'-NTs for vaccine development is also briefly discussed.

Publisher Correction: Mucosal vaccination with pili from Group A Streptococcus expressed on Lactococcus lactis generates protective immune responses.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Development of an opsonophagocytic killing assay for group a streptococcus.

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.

PilVax - a novel peptide delivery platform for the development of mucosal vaccines.

Peptide vaccines are an attractive strategy to engineer the induction of highly targeted immune responses and avoid potentially allergenic and/or reactogenic protein regions. However, peptides by themselves are often unstable and poorly immunogenic, necessitating the need for an adjuvant and a specialised delivery system. We have developed a novel peptide delivery platform (PilVax) that allows the presentation of a stabilised and highly amplified peptide as part of the group A streptococcus serotype M1 pilus structure (PilM1) on the surface of the non-pathogenic bacterium Lactococcus lactis. To show proof of concept, we have successfully inserted the model peptide Ova324-339 into 3 different loop regions of the backbone protein Spy0128, which resulted in the assembly of the pilus containing large numbers of peptide on the surface of L. lactis. Intranasal immunisation of mice with L. lactis PilM1-Ova generated measurable Ova-specific systemic and mucosal responses (IgA and IgG). Furthermore, we show that multiple peptides can be inserted into the PilVax platform and that peptides can also be incorporated into structurally similar, but antigenically different pilus structures. PilVax may be useful as a cost-effective platform for the development of peptide vaccines against a variety of important human pathogens.

Mucosal vaccination with pili from Group A Streptococcus expressed on Lactococcus lactis generates protective immune responses.

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.

Development of a high-throughput opsonophagocytic assay for the determination of functional antibody activity against Streptococcus pyogenes using bioluminescence.

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.

The Group A Streptococcus serotype M2 pilus plays a role in host cell adhesion and immune evasion.

Group A Streptococcus (GAS), or Streptococcus pyogenes, is a human pathogen that causes diseases ranging from skin and soft tissue infections to severe invasive diseases, such as toxic shock syndrome. Each GAS strain carries a particular pilus type encoded in the variable fibronectin-binding, collagen-binding, T antigen (FCT) genomic region. Here, we describe the functional analysis of the serotype M2 pilus encoded in the FCT-6 region. We found that, in contrast to other investigated GAS pili, the ancillary pilin 1 lacks adhesive properties. Instead, the backbone pilin is important for host cell adhesion and binds several host factors, including fibronectin and fibrinogen. Using a panel of recombinant pilus proteins, GAS gene deletion mutants and Lactococcus lactis gain-of-function mutants we show that, unlike other GAS pili, the FCT-6 pilus also contributes to immune evasion. This was demonstrated by a delay in blood clotting, increased intracellular survival of the bacteria in macrophages, higher bacterial survival rates in human whole blood and greater virulence in a Galleria mellonella infection model in the presence of fully assembled FCT-6 pili.