Mutation within the Streptococcus pyogenes (
group A; Strep A)
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
mutants, including M1T1 clonal strains (5448 and a
mutant generated through mouse passage designated 5448AP), to investigate the contribution of SLO to the pathogenesis of
mutant Strep A disease. Up-regulation of
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
-deletion mutant resulted in recovery of several
mutants within the 5448Δ
background. Passage also introduced mutations in non-
genes, but these were considered to have no impact on virulence. Although
-deficient mutants exhibited the characteristic
-controlled virulence factor upregulation, these mutants caused increased DC viability with reduced inflammatory cytokine production by infected DCs.
,
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
in the infecting strain (
mutant or wild-type) resulted in detection of DCs in the skin and attenuated virulence in a murine model of pyoderma.
-sufficient and -deficient
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.
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
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
and
-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.
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.
Peptides offer enormous promise as vaccines to prevent and protect against many infectious and noninfectious diseases. However, to date, limited vaccine efficacy has been reported and none have been ...licensed for human use. Innovative ways to enhance their immunogenicity are being tested, but rational sequence modification as a means to improve immune responsiveness has been neglected. Our objective was to establish a two-step generic protocol to modify defined amino acids of a helical peptide epitope to create a superior immunogen. Peptide variants of p145, a conserved helical peptide epitope from the M protein of
, were designed by exchanging one amino acid at a time, without altering their α-helical structure, which is required for correct antigenicity. The immunogenicities of new peptides were assessed in outbred mice. Vaccine efficacy was assessed in a skin challenge and invasive disease model. Out of 86 variants of p145, seven amino acid substitutions were selected and made the basis of the design for 18 new peptides. Of these, 13 were more immunogenic than p145; 7 induced Abs with significantly higher affinity for p145 than Abs induced by p145 itself; and 1 peptide induced more than 10,000-fold greater protection following challenge than the parent peptide. This peptide also only required a single immunization (compared with three immunizations with the parent peptide) to induce complete protection against invasive streptococcal disease. This study defines a strategy to rationally improve the immunogenicity of peptides and will have broad applicability to the development of vaccines for infectious and noninfectious diseases.
The present research had been attempted to formulate and characterize tocotrienols-rich naringenin nanoemulgel for topical application in chronic wound conditions associated with diabetes. In due ...course, different phases of the nanoemulsion were chosen based on the solubility study, where combination of Capryol 90 and tocotrienols, Solutol HS15, and Transcutol P were selected as oil, surfactant, and cosurfactant, respectively. The nanoemulsions were formulated using the spontaneous emulsification method. Subsequently, Carbopols were incorporated to develop corresponding nanoemulgels of the optimized nanoemulsions. Thermodynamically stable optimized nanoemulgels were evaluated for their globule size, polydispersity index (PDI), surface charge, viscosity, mucoadhesive property, spreadability, in vitro release and release mechanism. Further, increasing polymer concentration in the nanoemulgels was reflected with the increased mucoadhesive property with corresponding decrease in the release rate of the drug. The optimized nanoemulgel (NG1) consisted of uniform dispersion (PDI, 0.452 ± 0.03) of the nanometric globules (145.58 ± 12.5) of the dispersed phase, and negative surface charge (−21.1 ± 3.32 mV) with viscosity 297,600 cP and good spreadability. In vitro release of naringenin in phosphate buffer saline revealed a sustained release profile up to a maximum of 74.62 ± 4.54% from the formulated nanoemulgel (NG1) within the time-frame of 24 h. Alternatively, the release from the nanoemulsion was much higher (89.17 ± 2.87%), which might be due to lack of polymer coating on the dispersed oil droplets. Moreover, the in vitro release kinetics from the nanoemulgel followed the first-order release and Higuchi model with non-Fickian diffusion. Therefore, encouraging results in this research is evident in bringing a promising future in wound management, particularly associated with diabetes complications.
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•Thermodynamically stable naringenin-loaded tocotrienol-rich nanoemulgels were fabricated using spontaneous method.•Developed nanoemulgel possesses nanometric globule size with good spreadability.•Controlled in vitro release was obtained over a period of 24 h.•First-order release and Higuchi model with non-Fickian diffusion was established in the in vitro release kinetic profile.•This nanoemulgel could be a promising tool in the management of chronic wound condition.
The incidence of diabetes is increasing steeply; the number of diabetics has doubled over the past three decades. Surprisingly, the knowledge of type 3c diabetes mellitus (T3cDM) is still unclear to ...the researchers, scientist and medical practitioners, leading towards erroneous diagnosis, which is sometimes misdiagnosed as type 1 diabetes mellitus (T1DM), or more frequently type 2 diabetes mellitus (T2DM). This review is aimed to outline recent information on the etiology, pathophysiology, diagnostic procedures, and therapeutic management of T3cDM patients.
The literature related to T3cDM was thoroughly searched from the public domains and reviewed extensively to construct this article. Further, existing literature related to the other forms of diabetes is reviewed for projecting the differences among the different forms of diabetes. Detailed and updated information related to epidemiological evidence, risk factors, symptoms, diagnosis, pathogenesis and management is structured in this review.
T3cDM is often misdiagnosed as T2DM due to the insufficient knowledge differentiating between T2DM and T3cDM. The pathogenesis of T3cDM is explained which is often linked to the history of chronic pancreatitis, pancreatic cancer. Inflammation, and fibrosis in pancreatic tissue lead to damage both endocrine and exocrine functions, thus leading to insulin/glucagon insufficiency and pancreatic enzyme deficiency.
Future advancements should be accompanied by the establishment of a quick diagnostic tool through the understanding of potential biomarkers of the disease and newer treatments for better control of the diseased condition.
A successful vaccine needs to target multiple strains of an organism. Streptococcus pyogenes is an organism that utilizes antigenic strain variation as a successful defence mechanism to circumvent ...the host immune response. Despite numerous efforts, there is currently no vaccine available for this organism. Here we review and discuss the significant obstacles to vaccine development, with a focus on how cryptic epitopes may provide a strategy to circumvent the obstacles of antigenic variation.
We have developed a candidate vaccine to protect against multiple strains of Streptococcus pyogenes infections. The candidate vaccine contains two synthetic peptides derived from S. pyogenes ...proteins: the M-protein epitope, p*17 and the IL-8 degrading S. pyogenes Cell-Envelope Proteinase (SpyCEP) epitope, K4S2. In this study we utilise a rat autoimmune valvulitis model that displays both the cardiac and neurobehavioural pathology associated with post-streptococcal sequelae, to assess if the vaccine candidate antigens induce autoimmune complications and inflammatory pathology. Each antigen was conjugated to carrier protein diphtheria toxoid (DT) and independently assessed for potential to induce autoimmune pathology in female Lewis rats. Rats were administered three subcutaneous doses, and one intranasal dose over a four-week study with a two-week recovery period. A positive control group received recombinant S. pyogenes M5 (rM5) protein, and the negative control group received PBS. Rats that received rM5 developed significant cardiac and neurological pathologies. There was no evidence of these pathologies in the PBS control group, or the rats administered either P*17-DT or K4S2-DT. This study provides further preclinical evidence of the safety of the vaccine candidates p*17 and K4S2 and their appropriateness as candidates in human clinical trials.
Group A Streptococcus (GAS) skin infections are caused by a diverse array of strain types and are highly prevalent in disadvantaged populations. The role of strain-specific immunity in preventing GAS ...infections is poorly understood, representing a critical knowledge gap in vaccine development. A recent GAS murine challenge study showed evidence that sterilising strain-specific and enduring immunity required two skin infections by the same GAS strain within three weeks. This mechanism of developing enduring immunity may be a significant impediment to the accumulation of immunity in populations. We used an agent-based mathematical model of GAS transmission to investigate the epidemiological consequences of enduring strain-specific immunity developing only after two infections with the same strain within a specified interval. Accounting for uncertainty when correlating murine timeframes to humans, we varied this maximum inter-infection interval from 3 to 420 weeks to assess its impact on prevalence and strain diversity, and considered additional scenarios where no maximum inter-infection interval was specified. Model outputs were compared with longitudinal GAS surveillance observations from northern Australia, a region with endemic infection. We also assessed the likely impact of a targeted strain-specific multivalent vaccine in this context. Our model produced patterns of transmission consistent with observations when the maximum inter-infection interval for developing enduring immunity was 19 weeks. Our vaccine analysis suggests that the leading multivalent GAS vaccine may have limited impact on the prevalence of GAS in populations in northern Australia if strain-specific immunity requires repeated episodes of infection. Our results suggest that observed GAS epidemiology from disease endemic settings is consistent with enduring strain-specific immunity being dependent on repeated infections with the same strain, and provide additional motivation for relevant human studies to confirm the human immune response to GAS skin infection.
The potentials held by stimuli-responsive polymers in wound dressing have led to the present research in formulating a hydrogel base formulation with polymers having pH and thermo-sensitivity. Thus, ...hyaluronic acid (pH-sensitive polymer), and Pluronic F-127 (thermo-sensitive polymer) with hydroxypropyl methylcellulose (mucoadhesive polymer) were incorporated to obtain an
hydrogel containing gentamicin and naringenin (NAR). The optimization of the stimuli-responsive formulation was performed by the Box–Behnken statistical design to acquire variable parameters that influence the gelling temperature and viscosity. Thermo-gravimetric analysis, differential scanning calorimetry, and Fourier-transform infrared spectroscopy were performed to confirm the suitability of incorporating the selected polymers with drugs. The optimized formulations (blank and drug-loaded) were found to possess satisfactory characteristics of gelling temperatures (30–33°C), viscosities (174 ± 3 to 184 ± 4 cP), and mucoadhesive properties (0.29 ± 0.01 to 0.31 ± 0.01 N) with a spray diameter of 16.8 ± 1.4 to 18.9 ± 1.2 cm
to facilitate the application at the wound environment. The
drug release study depicted a sustained release profile over a time frame of 8 h with a cumulative release of 56.18 ± 4.59% NAR. The drug-containing
hydrogels showed superior potency by producing a larger zone of inhibition (2.03 ± 0.12 cm). Furthermore, a cytotoxicity study of the developed formulations in HaCaT cells revealed no toxicity of the drug-loaded formulations when compared to the blank hydrogel. These findings indicate the potential of the
hydrogel as an effective wound dressing for chronic wounds; however, additional investigation is needed for further implementation.
Group A Streptococcus (GAS) is an important human pathogen responsible for both superficial infections and invasive diseases. Autoimmune sequelae may occur upon repeated infection. For this reason, ...development of a vaccine against GAS represents a major challenge, since certain GAS components may trigger autoimmunity. We formulated three combination vaccines containing the following: (i) streptolysin O (SLO), interleukin 8 (IL-8) protease (Streptococcus pyogenes cell envelope proteinase SpyCEP), group A streptococcal C5a peptidase (SCPA), arginine deiminase (ADI), and trigger factor (TF); (ii) the conserved M-protein-derived J8 peptide conjugated to ADI; and (iii) group A carbohydrate lacking the N-acetylglucosamine side chain conjugated to ADI. We compared these combination vaccines to a "gold standard" for immunogenicity, full-length M1 protein. Vaccines were adjuvanted with alum, and mice were immunized on days 0, 21, and 28. On day 42, mice were challenged via cutaneous or subcutaneous routes. High-titer antigen-specific antibody responses with bactericidal activity were detected in mouse serum samples for all vaccine candidates. In comparison with sham-immunized mice, all vaccines afforded protection against cutaneous challenge. However, only full-length M1 protein provided protection in the subcutaneous invasive disease model.
This set of experiments demonstrates the inherent variability of mouse models for the characterization of GAS vaccine candidate protective efficacy. Such variability poses an important challenge for GAS vaccine development, as advancement of candidates to human clinical trials requires strong evidence of efficacy. This study highlights the need for an open discussion within the field regarding standardization of animal models for GAS vaccine development.
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