An efficient, chemically-defined semisynthetic lipid-adjuvanted nanoparticulate vaccine development system

A novel vaccine development platform that enables the site-specific conjugation of synthetic lipid adjuvants to recombinant proteins was produced. This technology facilitates the simple and efficient production of homogeneous, chemically-defined, semisynthetic lipoprotein vaccines. Using a polytope 'string-of-beads' approach, a synthetic gene incorporating seven Streptococcus pyogenes M protein strain-specific antigens, and a conserved M protein antigen (J14) was produced, expressed, and attached to a lipoamino acid based adjuvant (lipid core peptide; LCP). Nanoparticles (40 nm diameter) of an optimal size for stimulating antibody-mediated immunity were formed upon the addition of these lipoproteins to aqueous buffer (PBS). Systemic antigen-specific IgG antibodies were raised against all eight antigens in C57BL/6 J mice, without the need to formulate with additional adjuvant. These antibodies bound cell surface M proteins of S. pyogenes strains represented within the polytope sequence, with higher antibody levels observed where a dendritic cell targeting peptide (DCpep) was incorporated within the LCP adjuvant. Crown Copyright (C) 2013 Published by Elsevier Inc. All rights reserved

Mutual exclusivity of hyaluronan and hyaluronidase in invasive group A Streptococcus

A recent analysis of group A Streptococcus (GAS) invasive infections in Australia has shown a predominance of M4 GAS, a serotype recently reported to lack the antiphagocytic hyaluronic acid (HA) capsule. Here, we use molecular genetics and bioinformatics techniques to characterize 17 clinical M4 isolates associated with invasive disease in children during this recent epidemiology. All M4 isolates lacked HA capsule, and whole genome sequence analysis of two isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Conversely, M4 isolates possess a functional HA-degrading hyaluronate lyase (HylA) enzyme that is rendered nonfunctional in other GAS through a point mutation. Transformation with a plasmid expressing hasABC restored partial encapsulation in wild-type (WT) M4 GAS, and full encapsulation in an isogenic M4 mutant lacking HylA. However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection. Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition. These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen

Predicted Coverage and Immuno-Safety of a Recombinant C-Repeat Region Based Streptococcus pyogenes Vaccine Candidate

The C-terminal region of the M-protein of Streptococcus pyogenes is a major target for vaccine development. The major feature is the C-repeat region, consisting of 35-42 amino acid repeat units that display high but not perfect identity. SV1 is a S. pyogenes vaccine candidate that incorporates five 14mer amino acid sequences (called J14i variants) from differing C-repeat units in a single recombinant construct. Here we show that the J14i variants chosen for inclusion in SV1 are the most common variants in a dataset of 176 unique M-proteins. Murine antibodies raised against SV1 were shown to bind to each of the J14i variants present in SV1, as well as variants not present in the vaccine. Antibodies raised to the individual J14i variants were also shown to bind to multiple but different combinations of J14i variants, supporting the underlying rationale for the design of SV1. A Lewis Rat Model of valvulitis was then used to assess the capacity of SV1 to induce deleterious immune response associated with rheumatic heart disease. In this model, both SV1 and the M5 positive control protein were immunogenic. Neither of these antibodies were cross-reactive with cardiac myosin or collagen. Splenic T cells from SV1/CFA and SV1/alum immunized rats did not proliferate in response to cardiac myosin or collagen. Subsequent histological examination of heart tissue showed that 4 of 5 mice from the M5/CFA group had valvulitis and inflammatory cell infiltration into valvular tissue, whereas mice immunised with SV1/CFA, SV1/alum showed no sign of valvulitis. These results suggest that SV1 is a safe vaccine candidate that will elicit antibodies that recognise the vast majority of circulating GAS M-types

Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors

Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement and consequent phagocytosis. Several strains of GAS bind to human-specific complement inhibitors, C4b-binding protein (C4BP) and/or Factor H (FH), to curtail complement C3 (a critical opsonin) deposition. This results in diminished activation of phagocytes and clearance of GAS that may lead to the host being unable to limit the infection. Herein we describe the course of GAS infection in three human complement inhibitor transgenic (tg) mouse models that examined each inhibitor (human C4BP or FH) alone, or the two inhibitors together (C4BPxFH or 'double' tg). GAS infection with strains that bound C4BP and FH resulted in enhanced mortality in each of the three transgenic mouse models compared to infection in wild type mice. In addition, GAS manifested increased virulence in C4BPxFH mice: higher organism burdens and greater elevations of pro-inflammatory cytokines and they died earlier than single transgenic or wt controls. The effects of hu-C4BP and hu-FH were specific for GAS strains that bound these inhibitors because strains that did not bind the inhibitors showed reduced virulence in the 'double' tg mice compared to strains that did bind; mortality was also similar in wild-type and C4BPxFH mice infected by non-binding GAS. Our findings emphasize the importance of binding of complement inhibitors to GAS that results in impaired opsonization and phagocytic killing, which translates to enhanced virulence in a humanized whole animal model. This novel hu-C4BPxFH tg model may prove invaluable in studies of GAS pathogenesis and for developing vaccines and therapeutics that rely on human complement activation for efficacy

Characterisation of anchorless cell-surface proteins: novel candidates for a safe and universal group a streptococcal vaccine

Streptococcus pyogenes (group A streptococcus; GAS) causes ~700 million human infections each year, resulting in over 500,000 deaths. GAS can cause mild infections such as pharyngitis and impetigo, in addition to life threatening conditions including necrotising fasciitis, streptococcal toxic shock syndrome (STSS) and bacteremia. Repeated infection with GAS may result in the non-suppurative sequelae, acute rheumatic fever (ARF) and acute glomerulonephritis (APSGN). GAS remains sensitive to the antibiotic penicillin which can be administered as a means to treat infection or as prophylaxis. This strategy is utilised in regions with high GAS endemicity such as Indigenous populations living in northern Australia who suffer some of the highest rates of GAS auto-immune sequelae worldwide. However, issues with patient compliance and a growing concern over the possible emergence of resistant GAS strains may limit the usefulness of penicillin in the future. A vaccine capable of preventing GAS infection may be the only effective way to control and eliminate GAS infection and disease. The development of a commercial GAS vaccine is hampered by the occurrence of many unique GAS serotypes, antigenic variation within the same serotype, differences in geographical distribution of serotypes and the production of antibodies cross-reactive with human tissue that may lead to auto-immune disease. Several independent studies have documented a number of GAS cell wall-associated or secreted metabolic enzymes which contain neither N-terminal leader sequences nor C-terminal cell wall anchors. A proteomic analysis of serotype M1T1 GAS cell wall extracts was undertaken for the purpose of vaccine development. This approach catalogued 13 novel anchorless proteins and following a series of characterisation experiments identified two protective vaccine candidates, arginine deiminase (ADI) and trigger factor (TF). ADI and TF conferred protective efficacy in two murine models of GAS infection; systemic and subcutaneous. These surface-exposed antigens are expressed across multiple GAS serotypes exhibiting ≥ 99% amino acid sequence identity. Vaccine safety concerns are alleviated by the observation that these vaccine candidates lack human homologs, while sera from human populations suffering repeated GAS infections and high levels of auto-immune complications do not recognise these enzymes. In addition, anti-sera raised against ADI and TF does not react with human heart extract. Both ADI and TF warrant further investigation as GAS vaccine candidates for the prevention of GAS disease, which despite many decades of research trying to find a suitable vaccine, remains a major cause of morbidity and mortality worldwide

Mechanisms of group A Streptococcus resistance to reactive oxygen species

Streptococcus pyogenes, also known as group A Streptococcus (GAS), is an exclusively human Gram-positive bacterial pathogen ranked among the 'top 10' causes of infection-related deaths worldwide. GAS commonly causes benign and self-limiting epithelial infections (pharyngitis and impetigo), and less frequent severe invasive diseases (bacteremia, toxic shock syndrome and necrotizing fasciitis). Annually, GAS causes 700 million infections, including 1.8 million invasive infections with a mortality rate of 25%. In order to establish an infection, GAS must counteract the oxidative stress conditions generated by the release of reactive oxygen species (ROS) at the infection site by host immune cells such as neutrophils and monocytes. ROS are the highly reactive and toxic byproducts of oxygen metabolism, including hydrogen peroxide (H2O2), superoxide anion (O-2(center dot-)), hydroxyl radicals (OH center dot) and singlet oxygen (O-2(*)), which can damage bacterial nucleic acids, proteins and cell membranes. This review summarizes the enzymatic and regulatory mechanisms utilized by GAS to thwart ROS and survive under conditions of oxidative stress

Evaluation of novel Streptococcus pyogenes vaccine candidates incorporating multiple conserved sequences from the C-repeat region of the M-protein

A major challenge for Streptococcus pyogenes vaccine development is the identification of epitopes that confer protection from infection by multiple S. pyogenes M-types. Here we have identified and characterised the distribution of common variant sequences from individual repeat units of the C-repeat region (CRR) of M-proteins representing 77 different M-types. Three polyvalent fusion vaccine candidates (SV1, SV2 and SV3) incorporating the most common variants were subsequently expressed and purified, and demonstrated to be alpha-helical by Circular Dichroism (CD), a secondary conformational characteristic of the CRR in the M-protein. Antibodies raised against each of these constructs recognise M-proteins that vary in their CRR, and bind to the surface of multiple S. pyogenes isolates. Antibodies raised against SV1, containing five variant sequences, also kill heterologous S. pyogenes isolates in in vitro bactericidal assays. Further structural characterisation of this construct demonstrated the conformation of SV1 was stable at different pHs, and thermal unfolding of SV1 a reversible process. Our findings demonstrate that linkage of multiple variant sequences into a single recombinant construct overcomes the need to embed the variant sequences in foreign helix promoting flanking sequences for conformational stability, and demonstrates the viability of the polyvalent candidates as global S. pyogenes vaccine candidates

Pathogenesis of group A streptococcal infections

Group A Streptococcus (GAS; Streptococcus pyogenes) is a human pathogen which causes significant morbidity and mortality globally. GAS typically infects the throat and skin of the host, causing mild infections such as pharyngitis and impetigo, in addition to life threatening conditions including necrotizing fasciitis, streptococcal toxic shock syndrome (STSS), and bacteremia. Repeated infection with GAS may result in the non-suppurative sequelae, acute rheumatic fever, and acute glomerulonephritis. GAS remains sensitive to the antibiotic penicillin which can be administered as a means to treat infection or as prophylaxis. However, issues with patient compliance and a growing concern over the possible emergence of resistant GAS strains may limit the usefulness of antibiotics in the future. A vaccine capable of preventing GAS infection may be the only effective way to control and eliminate GAS infection and disease. [Discovery Medicine 13(72):329-342, may 2012