Streptococcal Immunity Is Constrained by Lack of Immunological Memory following a Single Episode of Pyoderma.

The immunobiology underlying the slow acquisition of skin immunity to group A streptococci (GAS), is not understood, but attributed to specific virulence factors impeding innate immunity and significant antigenic diversity of the type-specific M-protein, hindering acquired immunity. We used a number of epidemiologically distinct GAS strains to model the development of acquired immunity. We show that infection leads to antibody responses to the serotype-specific determinants on the M-protein and profound protective immunity; however, memory B cells do not develop and immunity is rapidly lost. Furthermore, antibodies do not develop to a conserved M-protein epitope that is able to induce immunity following vaccination. However, if re-infected with the same strain within three weeks, enduring immunity and memory B-cells (MBCs) to type-specific epitopes do develop. Such MBCs can adoptively transfer protection to naïve recipients. Thus, highly protective M-protein-specific MBCs may never develop following a single episode of pyoderma, contributing to the slow acquisition of immunity and to streptococcal endemicity in at-risk populations.

A synthetic M protein peptide synergizes with a CXC chemokine protease to induce vaccine-mediated protection against virulent streptococcal pyoderma and bacteremia.

Infections caused by Streptococcus pyogenes (group A Streptococcus [GAS]) are highly prevalent in the tropics, in developing countries, and in the Indigenous populations of developed countries. These infections and their sequelae are responsible for almost 500,000 lives lost prematurely each year. A synthetic peptide vaccine (J8-DT) from the conserved region of the M protein has shown efficacy against disease that follows i.p. inoculation of bacteria. By developing a murine model for infection that closely mimics human skin infection, we show that the vaccine can protect against pyoderma and subsequent bacteremia caused by multiple GAS strains, including strains endemic in Aboriginal communities in the Northern Territory of Australia. However, the vaccine was ineffective against a hypervirulent cluster of virulence responder/sensor mutant GAS strain; this correlated with the strain's ability to degrade CXC chemokines, thereby preventing neutrophil chemotaxis. By combining J8-DT with an inactive form of the streptococcal CXC protease, S. pyogenes cell envelope proteinase, we developed a combination vaccine that is highly effective in blocking CXC chemokine degradation and permits opsonic Abs to kill the bacteria. Mice receiving the combination vaccine were strongly protected against pyoderma and bacteremia, as evidenced by a 100-1000-fold reduction in bacterial burden following challenge. To our knowledge, a vaccine requiring Abs to target two independent virulence factors of an organism is unique.

Streptolysin O Deficiency in Streptococcus pyogenes M1T1 covR/S Mutant Strain Attenuates Virulence in In Vitro and In Vivo Infection Models.

Mutation within the Streptococcus pyogenes (Streptococcus group A; Strep A) covR/S regulatory system has been associated with a hypervirulent phenotype resulting from the upregulation of several virulence factors, including the pore-forming toxin, streptolysin O (SLO). In this study, we utilized a range of covR/S mutants, including M1T1 clonal strains (5448 and a covS mutant generated through mouse passage designated 5448AP), to investigate the contribution of SLO to the pathogenesis of covR/S mutant Strep A disease. Up-regulation of slo in 5448AP resulted in increased SLO-mediated hemolysis, decreased dendritic cell (DC) viability post coculture with Strep A, and increased production of tumor necrosis factor (TNF) and monocyte chemoattractant protein 1 (MCP-1) by DCs. Mouse passage of an isogenic 5448 slo-deletion mutant resulted in recovery of several covR/S mutants within the 5448Δslo background. Passage also introduced mutations in non-covR/S genes, but these were considered to have no impact on virulence. Although slo-deficient mutants exhibited the characteristic covR/S-controlled virulence factor upregulation, these mutants caused increased DC viability with reduced inflammatory cytokine production by infected DCs. In vivo, slo expression correlated with decreased DC numbers in infected murine skin and significant bacteremia by 3 days postinfection, with severe pathology at the infection site. Conversely, the absence of slo in the infecting strain (covR/S mutant or wild-type) resulted in detection of DCs in the skin and attenuated virulence in a murine model of pyoderma. slo-sufficient and -deficient covR/S mutants were susceptible to immune clearance mediated by a combination vaccine consisting of a conserved M protein peptide and a peptide from the CXC chemokine protease SpyCEP. IMPORTANCE Streptococcus pyogenes is responsible for significant numbers of invasive and noninvasive infections which cause significant morbidity and mortality globally. Strep A isolates with mutations in the covR/S system display greater propensity to cause severe invasive diseases, which are responsible for more than 163,000 deaths each year. This is due to the upregulation of virulence factors, including the pore-forming toxin streptolysin O. Utilizing covR/S and slo-knockout mutants, we investigated the role of SLO in virulence. We found that SLO alters interactions with host cell populations and increases Strep A viability at sterile sites of the host, such as the blood, and that its absence results in significantly less virulence. This work underscores the importance of SLO in Strep A virulence while highlighting the complex nature of Strep A pathogenesis. This improved insight into host-pathogen interactions will enable a better understanding of host immune evasion mechanisms and inform streptococcal vaccine development programs.

A Glycolipidated-liposomal peptide vaccine confers long-term mucosal protection against Streptococcus pyogenes via IL-17, macrophages and neutrophils.

Mucosally active subunit vaccines are an unmet clinical need due to lack of licensed immunostimulants suitable for vaccine antigens. Here, we show that intranasal administration of liposomes incorporating: the Streptococcus pyogenes peptide antigen, J8; diphtheria toxoid as a source of T cell help; and the immunostimulatory glycolipid, 3D(6-acyl) PHAD (PHAD), is able to induce long-lived humoral and cellular immunity. Mice genetically deficient in either mucosal antibodies or total antibodies are protected against S. pyogenes respiratory tract infection. Utilizing IL-17-deficient mice or depleting cellular subsets using antibodies, shows that the cellular responses encompassing, CD4+ T cells, IL-17, macrophages and neutrophils have important functions in vaccine-mediated mucosal immunity. Overall, these data demonstrate the utility of a mucosal vaccine platform to deliver multi-pronged protective responses against a highly virulent pathogen.

Combinatorial liposomal peptide vaccine induces IgA and confers protection against influenza virus and bacterial super-infection.

OBJECTIVES: The upper respiratory tract is the major entry site for Streptococcus pyogenes and influenza virus. Vaccine strategies that activate mucosal immunity could significantly reduce morbidity and mortality because of these pathogens. The severity of influenza is significantly greater if a streptococcal infection occurs during the viraemic period and generally viral infections complicated by a subsequent bacterial infection are known as super-infections. We describe an innovative vaccine strategy against influenza virus:S. pyogenes super-infection. Moreover, we provide the first description of a liposomal multi-pathogen-based platform that enables the incorporation of both viral and bacterial antigens into a vaccine and constitutes a transformative development. METHODS: Specifically, we have explored a vaccination strategy with biocompatible liposomes that express conserved streptococcal and influenza A virus B-cell epitopes on their surface and contain encapsulated diphtheria toxoid as a source of T-cell help. The vaccine is adjuvanted by inclusion of the synthetic analogue of monophosphoryl lipid A, 3D-PHAD. RESULTS: We observe that this vaccine construct induces an Immunoglobulin A (IgA) response in both mice and ferrets. Vaccination reduces viral load in ferrets from influenza challenge and protects mice from both pathogens. Notably, vaccination significantly reduces both mortality and morbidity associated with a super-infection. CONCLUSION: The vaccine design is modular and could be adapted to include B-cell epitopes from other mucosal pathogens where an IgA response is required for protection.

Antibodies to the conserved region of the M protein and a streptococcal superantigen cooperatively resolve toxic shock-like syndrome in HLA-humanized mice.

Invasive streptococcal disease (ISD) and toxic shock syndrome (STSS) result in over 160,000 deaths each year. We modelled these in HLA-transgenic mice infected with a clinically lethal isolate expressing Streptococcal pyrogenic exotoxin (Spe) C and demonstrate that both SpeC and streptococcal M protein, acting cooperatively, are required for disease. Vaccination with a conserved M protein peptide, J8, protects against STSS by causing a dramatic reduction in bacterial burden associated with the absence of SpeC and inflammatory cytokines in the blood. Furthermore, passive immunotherapy with antibodies to J8 quickly resolves established disease by clearing the infection and ablating the inflammatory activity of the M protein, which is further enhanced by addition of SpeC antibodies. Analysis of 77 recent isolates of Streptococcus pyogenes causing ISD, demonstrated that anti-J8 antibodies theoretically recognize at least 73, providing strong support for using antibodies to J8, with or without antibodies to SpeC, as a therapeutic approach.

Cellular interactions of covR/S mutant group A Streptococci.

Group A Streptococci (GAS) are responsible for a wide array of non-invasive and invasive diseases and varying immune sequelae with high rates of mortality and morbidity. GAS strains with a mutation in their covR/S regulatory system are hypervirulent with an increased capacity for causing invasive disease. covR/S mutants augment their virulence through the up-regulation of important virulence factors and target host immune surveillance primarily by inhibiting neutrophils. An in-depth understanding of the immunopathogenesis of covR/S mutants will facilitate the development of vaccine strategies and design. Ultimately, by targeting separate virulence mechanisms, multi-component vaccines may provide improved protective efficacy against hypervirulent GAS infections.

Skin infection boosts memory B-cells specific for a cryptic vaccine epitope of group A streptococcus and broadens the immune response to enhance vaccine efficacy.

Antigenic diversity of the M protein is a major constraint to the development of immunity to group A streptococcus (GAS). We demonstrate that a conserved cryptic epitope that is unrecognized by the host immune system following infection can protect mice following vaccination, and that immunity is strengthened and broadened following successive infections. The observation that infection can boost and broaden, but cannot prime immunity to a cryptic epitope, may be exploited for vaccines for other pathogens.

Novel platform technology for modular mucosal vaccine that protects against streptococcus.

The upper respiratory tract (URT) is the major entry site for human pathogens and strategies to activate this network could lead to new vaccines capable of preventing infection with many pathogens. Group A streptococcus (GAS) infections, causing rheumatic fever, rheumatic heart disease, and invasive disease, are responsible for substantial morbidity and mortality. We describe an innovative vaccine strategy to induce mucosal antibodies of significant magnitude against peptide antigens of GAS using a novel biocompatible liposomal platform technology. The approach is to encapsulate free diphtheria toxoid (DT), a standard vaccine antigen, within liposomes as a source of helper T-cell stimulation while lipidated peptide targets for B-cells are separately displayed on the liposome surface. As DT is not physically conjugated to the peptide, it is possible to develop modular epitopic constructs that simultaneously activate IgA-producing B-cells of different and complementary specificity and function that together neutralize distinct virulence factors. An inflammatory cellular immune response is also induced. The immune response provides profound protection against streptococcal infection in the URT. The study describes a new vaccine platform for humoral and cellular immunity applicable to the development of vaccines against multiple mucosal pathogens.