Preclinical safety and immunogenicity of Streptococcus pyogenes (Strep A) peptide vaccines.

We have developed two candidate vaccines to protect against multiple strains of Strep A infections. The candidates are combinatorial synthetic peptide vaccines composed of a M protein epitope (J8 or p*17) and a non-M protein epitope (K4S2). To enhance immunogenicity, each peptide is conjugated to the carrier protein CRM197 (CRM) and formulated with aluminium hydroxide adjuvant Alhydrogel (Alum) to make the final vaccines, J8-CRM + K4S2-CRM/Alum and p*17-CRM + K4S2-CRM/Alum. The safety and toxicity of each vaccine was assessed. Sprague Dawley rats were administered three intramuscular doses, over a six-week study with a 4-week recovery period. A control group received CRM only formulated with Alum (CRM/Alum). There was no evidence of systemic toxicity in the rats administered either vaccine. There was an associated increase in white blood cell, lymphocyte and monocyte counts, increased adrenal gland weights, adrenocortical hypertrophy, and increased severity of granulomatous inflammation at the sites of injection and the associated inguinal lymph nodes. These changes were considered non-adverse. All rats administered vaccine developed a robust and sustained immunological response. The absence of clinical toxicity and the development of an immunological response in the rats suggests that the vaccines are safe for use in a phase 1 clinical trial in healthy humans.

Epidemiological analysis of Group A Streptococcus infections in a hospital in Beijing, China.

Our study aimed to investigate the epidemiological and molecular characteristics of isolates collected from Group A Streptococcus (GAS) infections in children in Beijing China during the year 2019. Emm typing, superantigens, and erythromycin resistance genotypes were determined by PCR. Antimicrobial susceptibility testing was performed as recommended by Clinical Laboratory Standards Institute (CLSI). A total of 271 GAS isolates were collected. Thirteen different emm types, including 31 subtypes, were identified. The most prevalent emm types were emm12 (52.77%), emm1 (36.9%), emm3.1 (2.95%), and emm75.0 (2.95%). Two variant subtypes, STC36.0 and STG840.2, were identified. There was no difference in the portion of emm12 and emm1 isolates in scarlet fever, impetigo, and psoriasis. The majority of superantigens detected were smeZ (94.46%), speC (91.14%), and ssa (74.91%), followed by speH (56.46%), speI (45.76%), speJ (36.9%), and speA (34.32%). More scarlet fever isolates harbored speA (35.6%) and speJ (38.4%), more psoriasis isolates harbored speI (57.9%), and more impetigo isolates harbored ssa (89.7%). Isolates were universally susceptible to penicillin and resistant to erythromycin (94.83%). Moreover, 89.67% erythromycin resistance isolates harbored the ermB gene. The erythromycin resistance rate of the isolates from the three diseases was different. Scarlet fever is the common streptococcal infectious disease in dermatology. Emm12 and emm1 were the most prevalent emm types. The most prevalent superantigens detected were smeZ, spec, and ssa. There is association between diversity of superantigens and disease manifestation. Hence, continuous surveillance of GAS molecular epidemiological characterizations in different diseases is needed.

Molecular epidemiology and antimicrobial resistance of group a streptococcus recovered from patients in Beijing, China.

BACKGROUND: Group A streptococcus (GAS) is an important human pathogen responsible for a broad range of infections. Epidemiological surveillance has been crucial to detect changes in the geographical and temporal variation of the disease pattern. The objective of this study was to investigate the molecular epidemiological characteristics and antimicrobial resistance of GAS isolates from patients in Children's Hospital in Beijing. METHODS: From 2016 to 2017, pharyngeal swab samples were collected from the outpatients in Children's Hospital, Capital Institute of Pediatrics, who were diagnosed with scarlet fever. Antimicrobial susceptibility test was performed according to the distribution of conventional antibiotics and Clinical and Laboratory Standards Institute (CLSI) recommendations. The distribution of the macrolide-resistance genes (ermB, ermA, mefA), emm (M protein-coding gene) typing, and superantigens (SAg) gene profiling were examined by polymerase chain reaction (PCR). RESULTS: A total of 297 GAS isolates were collected. The susceptibility of the isolates to penicillin, ceftriaxone, and levofloxacin was 100%. The resistance rate to erythromycin and clindamycin was 98.3 and 96.6%, respectively. The dominant emm types were emm12 (65.32%), emm1 (27.61%), emm75 (2.69%), and emm89 (1.35%). Of the 297 isolates, 290 (97.64%) carried the ermB gene, and 5 (1.68%) carried the mefA gene, while none carried the ermA gene. The most common superantigen genes identified from GAS isolates were smeZ (96.97%), speC (92.59%), speG (91.58%), ssa (85.52%), speI (54.55%), speH (52.19%), and speA (34.34%). Isolates with the genotype emm1 possessed speA, speC, speG, speJ, speM, ssa, and smeZ, while emm12 possessed speC, speG, speH, speI, speM, ssa, and smeZ superantigens. CONCLUSIONS: The prevalent strain of GAS isolates in Beijing has a high resistance rate to macrolides; however, penicillin can still be the preferred antibiotic for treatment. Erythromycin resistance was predominantly mediated by ermB. The common emm types were emm12 and emm1. There was a correlation between emm and the superantigen gene. Thus, long-term monitoring and investigation of the emm types and superantigen genes of GAS prevalence are imperative.

Lancefield Whole Blood Killing Assay to Evaluate Vaccine Efficacy.

While the Lancefield whole blood killing assay is named after the renowned streptococcal researcher Rebecca Lancefield, the protocol was first described by Todd in 1927 (Br J Exp Pathol 8:1-5, 1927). Initially, the assay was used to identify novel Group A Streptococcal (GAS) serotypes through the supplementation of non-immune human blood (often from infants) with type-specific antisera prepared in rabbits (Lancefield, J Exp Med 106:525-544, 1957; Maxted, Br J Exp Pathol 37:415-422, 1956) and to demonstrate the impressive longevity of type-specific immunity in patients following invasive GAS infection (Lancefield, J Exp Med 110:271-292, 1959). The modern assay is routinely used to screen defined GAS mutants (Wessels, Bronze, Proc Natl Acad Sci U S A 91:12238-12242, 1994; Zinkernagel et al., Cell Host Microbe 4:170-178, 2008) or transposon libraries (Le Breton et al., Infect Immun 81:862-875, 2013) for enhanced susceptibility to opsonophagocytic killing or to screen vaccine antisera (Salehi et al., mSphere 3:e00617-e00618, 2018) or other serological preparations (Reglinski et al., Sci Rep 5:15825, 2015) for anti-streptococcal activity.

Mechanisms of microbial pathogenesis and the role of the skin microbiome in psoriasis: A review.

The pathogenesis of psoriasis may involve a breakdown of immune tolerance to cutaneous microorganisms. Psoriasis is associated with a higher incidence of Crohn disease and periodontitis, two diseases involving impaired tolerance and abnormal immune activation in response to intestinal and oral microbiota, respectively. In addition, guttate and chronic plaque psoriasis are associated with Streptococcus pyogenes colonization. The aim of this review is to characterize the microorganisms implicated in psoriasis by examining results of major association studies and possible mechanisms of pathogenesis. Although studies show relative increases in Streptococcus and Staphylococcus and decreases in Malassezia and Cutibacterium, they differ in methods of sampling and methods of microbial analysis. As such, no definitive associations between microbes and psoriasis have been found to date. It also remains unclear if changes in the microbiomal composition have a causal association with psoriasis or are simply a consequence of the inflammatory microenvironment. Techniques enabling strain-level analysis rather than species-level analysis of the skin microbiome are likely necessary to determine microbiomal signatures of psoriasis. Future investigations may lead to new diagnostic tests and novel treatments, such as probiotics or bacterial transplantation.

Protein adhesins as vaccine antigens for Group A Streptococcus.

Group A Streptococcus (GAS) is a globally important human pathogen that causes a broad spectrum of disease ranging from mild superficial infections to severe invasive diseases with high morbidity and mortality. Currently, there is no vaccine available for human use. GAS produces a vast array of virulence factors including multiple adhesin molecules. These mediate binding of the bacteria to host tissues and are essential in the initial phases of infection. Prophylactic vaccination with adhesins is a promising vaccine strategy and many GAS adhesins are currently in development as vaccine candidates. The most advanced candidates, having entered clinical trials, are based on the M protein, while components of the pilus and a number of fibronectin-binding proteins are in pre-clinical development. Adhesin-based vaccines aim to induce protective immunity via two main mechanisms: neutralisation where adhesin-specific antibodies block the ability of the adhesin to bind to host tissue and opsonisation in which adhesin-specific antibodies tag the GAS bacteria for phagocytosis. This review summarises our current knowledge of GAS adhesins and their structural features in the context of vaccine development.

Immunization with Streptococcal Heme Binding Protein (Shp) Protects Mice Against Group A Streptococcus Infection.

Streptococcal heme binding protein (Shp) is a surface protein of the heme acquisition system that is an essential iron nutrient in Group A Streptococcus (GAS). Here, we tested whether Shp immunization protects mice from subcutaneous infection. Mice were immunized subcutaneously with recombinant Shp and then challenged with GAS. The protective effects against GAS challenge were evaluated two weeks after the last immunization. Immunization with Shp elicited a robust IgG response, resulting in high anti-Shp IgG titers in the serum. Immunized mice had a higher survival rate and smaller skin lesions than adjuvant control mice. Furthermore, immunized mice had lower GAS numbers at the skin lesions and in the liver, spleen and lung. Histological analysis with Gram staining showed that GAS invaded the surrounding area of the inoculation sites in the skin in control mice, but not in immunized mice. Thus, Shp immunization enhances GAS clearance and reduces GAS skin invasion and systemic dissemination. These findings indicate that Shp is a protective antigen.

Preclinical safety study of a recombinant Streptococcus pyogenes vaccine formulated with aluminum adjuvant.

A recombinant vaccine composed of a fusion protein formulated with aluminum hydroxide adjuvant is under development for protection against diseases caused by Streptococcus pyogenes. The safety and local reactogenicity of the vaccine was assessed by a comprehensive series of clinical, pathologic and immunologic tests in preclinical experiments. Outbred mice received three intramuscular injections of 1/5th of the human dose (0.1 ml) and rabbits received two injections of the full human dose. Control groups received adjuvant or protein antigen. The vaccine did not cause clinical evidence of systemic toxicity in mice or rabbits. There was a transient increase of peripheral blood neutrophils after the third vaccination of mice. In addition, the concentration of acute phase proteins serum amyloid A and haptoglobin was significantly increased 1 day after injection of the vaccine in mice. There was mild transient swelling and erythema of the injection site in both mice and rabbits. Treatment-related pathology was limited to inflammation at the injection site and accumulation of adjuvant-containing macrophages in the draining lymph nodes. In conclusion, the absence of clinical toxicity in two animal species suggest that the vaccine is safe for use in a phase I human clinical trial. Copyright © 2016 John Wiley & Sons, Ltd.

Preclinical immunogenicity and safety of a Group A streptococcal M protein-based vaccine candidate.

Streptococcus pyogenes (group A streptococcus, GAS) causes a wide range of clinical manifestations ranging from mild self-limiting pyoderma to invasive diseases such as sepsis. Also of concern are the post-infectious immune-mediated diseases including rheumatic heart disease. The development of a vaccine against GAS would have a large health impact on populations at risk of these diseases. However, there is a lack of suitable models for the safety evaluation of vaccines with respect to post-infectious complications. We have utilized the Lewis Rat model for cardiac valvulitis to evaluate the safety of the J8-DT vaccine formulation in parallel with a rabbit toxicology study. These studies demonstrated that the vaccine did not induce abnormal pathology. We also show that in mice the vaccine is highly immunogenic but that 3 doses are required to induce protection from a GAS skin challenge even though 2 doses are sufficient to induce a high antibody titer.

Structure-activity relationship of lipid core peptide-based Group A Streptococcus vaccine candidates.

Infection with Group A Streptococcus (GAS) can result in a range of different illnesses, some of which are fatal. Currently, our efforts to develop a vaccine against GAS focuses on the lipid core peptide (LCP) system, a subunit vaccine containing a lipoamino acid (LAA) moiety which allows the stimulation of systemic antibody activity. In the present study, a peptide (J14) representing the B-cell epitope from the GAS M protein was incorporated alongside a universal T-helper epitope (P25) in four LCP constructs of different spatial orientation or LAA lengths. Through structure-activity studies, it was discovered that while the alteration of the LCP orientation had a weaker effect on immunostimulation, increasing the LAA side chain length within the construct increased antibody responses in murine models. Furthermore, the mice immunised with the lead LCP construct were also able to maintain antibody activity throughout the course of five months. These findings highlight the importance of LAA moieties in the development of intranasal peptide vaccines and confirmed that its side chain length has an effect on the immunogenicity of the structure.

Streptococcus pyogenes arginine and citrulline catabolism promotes infection and modulates innate immunity.

A bacterium's ability to acquire nutrients from its host during infection is an essential component of pathogenesis. For the Gram-positive pathogen Streptococcus pyogenes, catabolism of the amino acid arginine via the arginine deiminase (ADI) pathway supplements energy production and provides protection against acid stress in vitro. Its expression is enhanced in murine models of infection, suggesting an important role in vivo. To gain insight into the function of the ADI pathway in pathogenesis, the virulence of mutants defective in each of its enzymes was examined. Mutants unable to use arginine (ΔArcA) or citrulline (ΔArcB) were attenuated for carriage in a murine model of asymptomatic mucosal colonization. However, in a murine model of inflammatory infection of cutaneous tissue, the ΔArcA mutant was attenuated but the ΔArcB mutant was hyperattenuated, revealing an unexpected tissue-specific role for citrulline metabolism in pathogenesis. When mice defective for the arginine-dependent production of nitric oxide (iNOS(-/-)) were infected with the ΔArcA mutant, cutaneous virulence was rescued, demonstrating that the ability of S. pyogenes to utilize arginine was dispensable in the absence of nitric oxide-mediated innate immunity. This work demonstrates the importance of arginine and citrulline catabolism and suggests a novel mechanism of virulence by which S. pyogenes uses its metabolism to modulate innate immunity through depletion of an essential host nutrient.

[Multifocal inflammatory syndrome after invasive infection due to an M1 strain of Streptococcus pyogenes]. / Manifestations inflammatoires réactionnelles multifocales après infection invasive à Streptococcus pyogenes de type M1.

We report on a case of Streptococcus pyogenes invasive disease with toxic shock syndrome due to an M1 strain producing SpeA and SmeZ superantigenic toxins. Post-streptococcal sequelae included several episodes of reactive arthritis and orchitis whose outcome was favorable with corticosteroid therapy. Invasive streptococcal infections are increasingly reported and may associate septic, toxinic, and immunological diseases. High-grade systemic inflammation may induce nonsuppurative complications and autoimmune diseases by molecular mimicry. Among them, reactive arthritis has been recognized as a separate entity from acute rheumatic fever and post-streptococcal orchitis has not been described before. Treatment should be quickly started and should be effective on the etiologic agent but also on its toxins due to the severity of the invasive infections associated with the spread of highly virulent bacterial clones and the potential development of multifocal nonsuppurative sequelae.

Impact of rapid antigen detection testing on antibiotic prescription in acute pharyngitis in adults. FARINGOCAT STUDY: a multicentric randomized controlled trial.

BACKGROUND: Acute pharyngitis is one of the most frequent consultations to the general practitioner and in most of the cases an antibiotic is prescribed in primary care in Spain. Bacterial etiology, mainly by group A beta-hemolytic streptococcus (GABHS), accounts for 10-20% of all these infections in adults. The purpose of this study is to assess the impact of rapid antigen detection testing (RADT) to identify GABHS in acute pharyngitis on the utilization of antibiotics in primary care. METHODS/DESIGN: Multicentric randomized controlled trial in which antibiotic prescription between two groups of patients with acute pharyngitis will be compared. The trial will include two arms, a control and an intervention group in which RADT will be performed. The primary outcome measure will be the proportion of inappropriate antibiotic prescription in each group. Two hundred seventy-six patients are required to detect a reduction in antibiotic prescription from 85% in the control group to 75% in the intervention group with a power of 90% and a level of significance of 5%. Secondary outcome measures will be specific antibiotic treatment, antibiotic resistance rates, secondary effects, days without working, medical visits during the first month and patient satisfaction. DISCUSSION: The implementation of RADT would allow a more rational use of antibiotics and would prevent adverse effects of antibiotics, emergence of antibiotic resistance and the growth of inefficient health expenses.

A vaccine against S. pyogenes: design and experimental immune response.

Streptococcus pyogenes causes severe invasive infections: the post-streptococcal sequelae of acute rheumatic fever (RF) and rheumatic heart disease (RHD), acute glomerulonephritis, and uncomplicated pharyngitis and pyoderma. Efforts to produce a vaccine against S. pyogenes began several decades ago, and different models have been proposed. Here, we describe the methodology used in the development of a new vaccine model, consisting of both T and B protective epitopes constructed as synthetic peptides and recombinant proteins. Two adjuvants were tested in an experimental inbred mouse model: a classical Freund's adjuvant and a new adjuvant (AFCo1) that induces mucosal immune responses and is obtained by calcium precipitation of a proteoliposome derived from the outer membrane of Neisseria meningitides B. The StreptInCor vaccine epitope co-administrated with AFCo1 adjuvant induced mucosal (IgA) and systemic (IgG) antibodies as preferential Th1-mediated immune responses. No autoimmune reactions were observed, suggesting that the vaccine epitope is safe.

Synthesis of a Streptococcus pyogenes vaccine candidate based on the M protein PL1 epitope.

Group A streptococcus is a Gram-positive bacteria that causes a range of infectious diseases. Targeting the bacteria, a new self-adjuvanting vaccine candidate, incorporating a carbohydrate carrier and an amino acid-based adjuvant, was synthesised utilising carbohydrate chemistry and solid-phase peptide synthesis procedures. Characterisation of the candidate was achieved using reverse-phase HPLC and electrospray ionisation mass spectrometry. The successful synthesis and characterisation of the vaccine candidate may contribute to the discovery of a therapeutically and clinically viable vaccine against group A streptococcus.

Extract of Pelargonium sidoides (EPs 7630) displays anti-infective properties by enhanced phagocytosis and differential modulation of host-bacteria interactions.

EPs 7630 is an aqueous-ethanolic extract of the roots of PELARGONIUM SIDOIDES that displays well-documented benefits in the treatment of upper respiratory tract infections (URTI). IN VITRO and animal investigations have revealed various anti-infective properties of EPs 7630. The present review sums up recently published IN VITRO investigations that have shown positive effects on the activity of human peripheral blood phagocytes (PBP) and differential modulation of the interactions between group A streptococci and the host's epithelial barrier.

Structure-activity relationship of a series of synthetic lipopeptide self-adjuvanting group a streptococcal vaccine candidates.

The development of 16 self-adjuvanting group A streptococcal vaccine candidates, composed of (i) a universal helper T-cell epitope (P25), (ii) a target GAS B-cell epitope (J14), and (iii) a lipid moiety, is described. Systemic J14-specific IgG antibodies were detected following subcutaneous immunization of BALB/c (H-2 (d)) mice with each construct without the need for an additional adjuvant. The effect of changing the order of P25, J14, and lipid moiety attachment or incorporation of P25 and J14 into a lipid-core peptide system on antibody titers was assessed. The point of lipid moiety attachment had the greatest influence on systemic J14-specific IgG antibody titers. Overall, the best vaccines featured a C-terminal lipid moiety, conjugated through a lysine residue to P25 at the N-terminus, and J14 on the lysine side chain.

Method for the synthesis of multi-epitopic Streptococcus pyogenes lipopeptide vaccines using native chemical ligation.

The aim of this study was to investigate methods for the synthesis of highly pure, well-characterized analogues of the lipid core peptide (LCP) system. Difficulties synthesizing and purifying conventional LCP systems have led to the requirement for a technique to produce highly pure, LCP-based vaccines for potential use in human clinical trials. The current study describes methods for the attachment of lipophilic adjuvants onto multi-epitopic peptide vaccines. Described is the synthesis, using native chemical ligation, of a highly pure, tri-epitopic, group A streptococcal (GAS) lipopeptide vaccine candidate. Intranasal immunization of the described tri-epitopic GAS lipopeptide with the mucosal adjuvant cholera toxin B subunit induced high serum IgG antibody titers specific for each of the incorporated peptide epitopes.

StreptAvax (ID Biomedical).

ID Biomedical, under license from the University of Tennessee, is developing StreptAvax, a potential subunit vaccine against group A streptococcal infection. By January 2005, analysis of data from phase II clinical trials conducted in adults was completed. Pediatric trials are not expected to begin before 2007.

Identification and assessment of new vaccine candidates for group A streptococcal infections.

Group A Streptococcus (GAS) is a human-specific pathogen responsible for a wide variety of human diseases. Numerous GAS surface antigens interact with the human immune system and only some of these proteins have been studied in depth. A few of these may elicit protective response against GAS infection. In this study, we have used an in silico approach to identify antigenic peptides from GAS surface proteins. Putative GAS surface proteins from the M1 GAS genome were identified by the presence on LPxTG cell-wall anchoring motif and an export signal sequence. This technique identified 17 proteins of known or putative function, and another 11 which do not have known homologues. Peptides derived from predicted antigenic sequences near the amino terminus of six of these proteins, and another seven peptides derived from the two known surface proteins, GRAB and MtsA, were conjugated to keyhole lymphocyanin (KLH), and investigated for their capacity to induce opsonic antibody responses in outbred Quackenbush mice. All peptide-KLH antisera demonstrated opsonic capacity against both 88/30 and M1 GAS. However, KLH sera alone was also able to induce opsonic antibodies, suggesting that anti-KLH antibodies contributed to the opsonisation seen in the peptide-KLH antisera. KLH is therefore a promising carrier molecule for potential GAS peptide vaccines.